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TW201518322A - Novel anti-CD38 antibody for the treatment of cancer - Google Patents

Novel anti-CD38 antibody for the treatment of cancer Download PDF

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Publication number
TW201518322A
TW201518322A TW103126457A TW103126457A TW201518322A TW 201518322 A TW201518322 A TW 201518322A TW 103126457 A TW103126457 A TW 103126457A TW 103126457 A TW103126457 A TW 103126457A TW 201518322 A TW201518322 A TW 201518322A
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TW
Taiwan
Prior art keywords
antibody
cells
binding fragment
methyl
group
Prior art date
Application number
TW103126457A
Other languages
Chinese (zh)
Other versions
TWI609884B (en
Inventor
Peter U Park
Laura M Bartle
Anna Skaletskaya
Viktor S Golmakher
Daniel Tavares
Jutta Deckert
Vincent Mikol
Veronique Blanc
Original Assignee
Sanofi Aventis
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Application filed by Sanofi Aventis filed Critical Sanofi Aventis
Publication of TW201518322A publication Critical patent/TW201518322A/en
Application granted granted Critical
Publication of TWI609884B publication Critical patent/TWI609884B/en

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Abstract

本發明提供特異性結合CD38之抗體、人類化抗體、表面重塑(resurfaced)抗體、抗體片段、衍生抗體、及其與細胞毒性劑之結合物，其能夠經由細胞凋亡(apoptosis)、抗體依賴性細胞介導之細胞毒性(ADCC)及/或補體依賴性細胞毒性(CDC)殺死CD38+細胞。該等抗體及其片段可用於治療表現CD38蛋白之腫瘤，諸如多發性骨髓瘤、慢性淋巴細胞性白血病、慢性骨髓性白血病、急性骨髓性白血病或急性淋巴細胞性白血病；或治療自體免疫及發炎疾病，諸如全身性狼瘡、類風濕性關節炎、多發性硬化症、紅斑及哮喘。該等衍生抗體可用於診斷CD38表現量升高之腫瘤及使該等腫瘤成像。亦提供包含細胞結合劑及細胞毒性劑之細胞毒性結合物，包含該結合物之治療組合物，使用該結合物抑制細胞生長及治療疾病之方法，及包含該細胞毒性結合物之套組。特別言之，該細胞結合劑為識別及結合CD38蛋白之單株抗體及其抗原決定基(epitope)結合片段。 The present invention provides an antibody, a humanized antibody, a surface remodeling antibody, an antibody fragment, a derivative antibody, and a conjugate thereof with a cytotoxic agent which specifically bind to CD38, which are capable of apoptosis-dependent, antibody-dependent Sex cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) kills CD38+ cells. Such antibodies and fragments thereof can be used to treat tumors exhibiting CD38 protein, such as multiple myeloma, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia or acute lymphocytic leukemia; or treatment of autoimmune and inflammatory Diseases such as systemic lupus, rheumatoid arthritis, multiple sclerosis, erythema and asthma. Such derivatized antibodies can be used to diagnose and image tumors with elevated CD38 expression. Also provided are cytotoxic conjugates comprising a cell binding agent and a cytotoxic agent, a therapeutic composition comprising the conjugate, a method of inhibiting cell growth and treating a disease using the conjugate, and a kit comprising the cytotoxic conjugate. In particular, the cell binding agent is a monoclonal antibody that recognizes and binds to the CD38 protein and an epitope binding fragment thereof.

Description

用於治療癌症之新穎抗-CD38抗體 Novel anti-CD38 antibody for the treatment of cancer

CD38為具有長C末端細胞外域及短N末端細胞質域之45kD II型跨膜糖蛋白。CD38蛋白為可催化NAD+轉化成環ADP-核糖(cADPR)且亦使cADPR水解成ADP-核糖之雙功能胞外酶。在個體發生期間，CD38出現於CD34+定型幹細胞上及淋巴細胞、紅血球及骨髓細胞之系定型祖細胞上。CD38表現主要在淋巴系中持續，並且在T細胞及B細胞發育之不同階段表現量不同。 CD38 is a 45kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. The CD38 protein is a bifunctional extracellular enzyme that catalyzes the conversion of NAD + to cyclic ADP-ribose (cADPR) and also hydrolyzes cADPR to ADP-ribose. During the onset of the individual, CD38 appears on CD34 + committed stem cells and on committed lymphoblastic cells of lymphocytes, red blood cells and bone marrow cells. CD38 expression persists primarily in the lymphoid system and varies in the amount of T cell and B cell development at different stages.

CD38在許多造血惡性疾病中及來源於各種造血惡性疾病之細胞株中上調，該等造血惡性疾病包括非霍奇金氏淋巴瘤(non-Hodgkin's lymphoma，NHL)、伯基特氏淋巴瘤(Burkitt's lymphoma，BL)、多發性骨髓瘤(MM)、慢性B淋巴細胞性白血病(B-CLL)、急性B及T淋巴細胞性白血病(ALL)、T細胞淋巴瘤(TCL)、急性骨髓性白血病(AML)、毛細胞白血病(HCL)、霍奇金氏淋巴瘤(Hodgkin's Lymphoma，HL)及慢性骨髓性白血病(CML)。另一方面，大多數造血系統之未分化多功能幹細胞為CD38-。造血惡性疾病中之CD38表現及其與疾病進程之相關性使得CD38成為抗體療法之受關注標靶。 CD38 is up-regulated in many hematopoietic malignancies and in cell lines derived from various hematopoietic malignancies including non-Hodgkin's lymphoma (NHL) and Burkitt's lymphoma (Burkitt's). Lymphoma, BL), multiple myeloma (MM), chronic B lymphocytic leukemia (B-CLL), acute B and T lymphocytic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia ( AML), hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myelogenous leukemia (CML). On the other hand, the undifferentiated pluripotent stem cells of most hematopoietic systems are CD38 - . CD38 expression in hematopoietic malignancies and its association with disease progression make CD38 a target of antibody therapy.

已報導CD38與Ca2+動員有關(M.Morra等人，1998,FASEB J.,12：581-592；M.T.Zilber等人，2000,Proc Natl Acad Sci U S A,97：2840-2845)且與在淋巴細胞及骨髓細胞或細胞株中經由許多信號轉導分子 (包括磷脂酶C-γ、ZAP-70、syk及c-cbl)之酪胺酸磷酸化的信號轉導有關(A.Funaro等人，1993,Eur J Immunol,23：2407-2411；M.Morra等人，1998,FASEB J.,12：581-592；A.Funaro等人，1990,J Immunol,145：2390-2396；M.Zubiaur等人，1997,J Immunol,159：193-205；S.Deaglio等人，2003,Blood 102：2146-2155；E.Todisco等人，2000,Blood,95：535-542；M.Konopleva等人，1998,J Immunol,161：4702-4708；M.T.Zilber等人，2000,Proc Natl Acad Sci U S A,97：2840-2845；A.Kitanaka等人，1997,J Immunol,159：184-192；A.Kitanaka等人，1999,J Immunol,162：1952-1958；R.Mallone等人，2001,Int Immunol,13：397-409)。基於此等觀察資料，提出CD38為淋巴細胞及骨髓細胞在其正常發育期間成熟及活化中之重要信號轉導分子。 It has been reported that CD38 is involved in Ca 2+ mobilization (M. Morra et al., 1998, FASEB J. , 12 : 581-592; MTZilber et al., 2000, Proc Natl Acad Sci USA , 97 : 2840-2845) and with lymphoid Signal transduction of tyrosine phosphorylation by many signal transduction molecules, including phospholipases C-γ, ZAP-70, syk, and c-cbl, in cells and bone marrow cells or cell lines (A. Funaro et al., 1993, Eur J Immunol , 23 : 2407-2411; M. Morra et al., 1998, FASEB J. , 12 : 581-592; A. Funaro et al., 1990, J Immunol , 145 : 2390-2396; M. Zubiaur Et al, 1997, J Immunol , 159 : 193-205; S. Deaglio et al, 2003, Blood 102 : 2146-2155; E. Todisco et al, 2000, Blood , 95 : 535-542; M. Konopleva et al. , 1998, J Immunol , 161 : 4702-4708; MTZilber et al, 2000, Proc Natl Acad Sci USA , 97 : 2840-2845; A. Kitanaka et al, 1997, J Immunol , 159 : 184-192; A. Kitanaka Et al, 1999, J Immunol , 162 :1952-1958; R. Mallone et al, 2001, Int Immunol , 13 :397-409). Based on these observations, it is proposed that CD38 is an important signal transduction molecule for the maturation and activation of lymphocytes and bone marrow cells during their normal development.

CD38在信號轉導及造血中之確切作用仍不清楚，此尤其係由於大多數此等信號轉導研究係使用異位過度表現CD38及抗-CD38單株抗體(其為非生理性配位子)之細胞株。由於CD38蛋白具有產生cADPR(一種可誘發Ca2+動員之分子)之酶活性(H.C.Lee等人，1989,J Biol Chem,264：1608-1615；H.C.Lee及R.Aarhus,1991,Cell Regul,2：203-209)，因此已提出CD38與單株抗體接合藉由增加cADPR產生而觸發淋巴細胞中之Ca2+動員及信號轉導(H.C.Lee等人，1997,Adv Exp Med Biol,419：411-419)。與此假說相反，CD38蛋白之截斷及點突變分析顯示其細胞質尾或其酶活性均非由抗-CD38抗體介導之信號轉導所必需(A.Kitanaka等人，1999,J Immunol, 162：1952-1958；F.E.Lund等人，1999,J Immunol,162：2693-2702；S.Hoshino等人，1997,J Immunol,158,741-747)。 The exact role of CD38 in signal transduction and hematopoiesis remains unclear, especially since most of these signal transduction studies use ectopic overexpression of CD38 and anti-CD38 monoclonal antibodies (which are non-physiological ligands). ) cell line. Since CD38 protein has an enzymatic activity that produces cADPR, a molecule that induces Ca 2+ mobilization (HCLee et al., 1989, J Biol Chem , 264 : 1608-1615; HCLee and R. Aarhus, 1991, Cell Regul , 2 : 203-209), therefore, it has been proposed that CD38 binding to monoclonal antibodies triggers Ca 2+ mobilization and signal transduction in lymphocytes by increasing cADPR production (HCLee et al., 1997, Adv Exp Med Biol , 419 :411-419). ). Contrary to this hypothesis, truncation and point mutation analysis of CD38 protein revealed that its cytoplasmic tail or its enzymatic activity is not required for signal transduction mediated by anti-CD38 antibodies (A. Kitanaka et al., 1999, J Immunol, 162 : 1952-1958; FELund et al, 1999, J Immunol , 162 : 2693-2702; S. Hoshino et al, 1997, J Immunol , 158 , 741-747).

對於CD38功能之最佳證據來自CD38-/-基因剔除小鼠，該等小鼠由於樹突狀細胞遷移中之缺陷而在先天免疫力方面具有缺陷且具有降低之T細胞依賴性體液反應(S.Partida-Sanchez等人，2004,Immunity, 20：279-291；S.Partida-Sanchez等人，2001,Nat Med,7：1209-1216)。然而，尚不清楚小鼠中之CD38功能是否與人類中之CD38功能相同，因為造血期間之CD38表現模式在人類與小鼠之間大不相同：a)不同於人類之未成熟祖幹細胞，小鼠之類似祖幹細胞具較高CD38表現量(T.D.Randall等人，1996,Blood,87：4057-4067；R.N.Dagher等人，1998,Biol Blood Marrow Transplant,4：69-74)；b)在人類B細胞發育期間，高CD38表現量係見於生髮中心B細胞及漿細胞中(F.M.Uckun,1990,Blood,76：1908-1923；M.Kumagai等人，1995,J Exp Med,181：1101-1110)，而在小鼠中，相應細胞中之CD38表現量較低(A.M.Oliver等人，1997,J Immunol,158：1108-1115；A.Ridderstad及D.M.Tarlinton 1998,J Immunol,160：4688-4695)。 The best evidence for CD38 function comes from CD38 - / - knockout mice, which are defective in innate immunity due to defects in dendritic cell migration and have a reduced T cell-dependent humoral response (S .Partida-Sanchez et al, 2004, Immunity , 20 : 279-291; S. Partida-Sanchez et al, 2001, Nat Med , 7 : 1209-1216). However, it is unclear whether CD38 function in mice is functionally equivalent to CD38 in humans, since the pattern of CD38 expression during hematopoiesis is quite different between humans and mice: a) different from human immature progenitor stem cells, small Murine-like progenitor stem cells have higher CD38 expression (TDRandall et al, 1996, Blood , 87 : 4057-4067; RNDagher et al, 1998, Biol Blood Marrow Transplant , 4 : 69-74); b) in human B cells During development, high CD38 expression is found in germinal center B cells and plasma cells (FMUckun, 1990, Blood , 76 : 1908-1923; M. Kumagai et al, 1995, J Exp Med , 181 : 1101-1110). In mice, the amount of CD38 expression in the corresponding cells is low (AMO Liver et al, 1997, J Immunol , 158 : 1108-1115; A. Ridderstad and DMTarlinton 1998, J Immunol , 160 : 4688-4695).

文獻中已描述數種對各種腫瘤細胞及細胞株具有不同增殖特性之抗人類CD38抗體。舉例而言，具有小鼠Fab及人類IgG1 Fc之嵌合OKT10抗體在來自MM患者或正常個體之周邊血液單核效應細胞存在下介導極有效對抗淋巴瘤細胞之抗體依賴性細胞介導之細胞毒性(ADCC)(F.K.Stevenson等人1991,Blood,77：1071-1079)。已顯示抗-CD38抗體AT13/5之CDR移植人類化型式具有對抗CD38-陽性細胞株之有效ADCC活性(US 09/797,941 A1)。已顯示人類單株抗-CD38抗體經由ADCC及/或補體依賴性細胞毒性(CDC)介導活體外殺死CD38-陽性細胞株，且延緩負載MM細胞株RPMI-8226之SCID小鼠中之腫瘤生長(WO2005/103083 A2)。另一方面，數種抗-CD38抗體，IB4、SUN-4B7及OKT10(但非IB6、AT1或AT2)，誘發來自正常個體之周邊血液單核細胞(PBMC)增殖(C.M.Ausiello等人2000,Tissue Antigens,56：539-547)。 Several anti-human CD38 antibodies with different proliferative properties against various tumor cells and cell lines have been described in the literature. For example, a chimeric OKT10 antibody with mouse Fab and human IgG1 Fc mediates antibody-dependent cell-mediated cells that are highly potent against lymphoma cells in the presence of peripheral blood mononuclear effector cells from MM patients or normal individuals. Toxicity (ADCC) (FK Stevenson et al. 1991, Blood , 77 : 1071-1079). The CDR-grafted humanized version of the anti-CD38 antibody AT13/5 has been shown to have potent ADCC activity against CD38-positive cell lines (US 09/797,941 A1). Human monoclonal anti-CD38 antibodies have been shown to mediate CD38-positive cell lines in vitro via ADCC and/or complement dependent cytotoxicity (CDC) and delay tumors in SCID mice bearing MM cell line RPMI-8226 Growth (WO2005/103083 A2). On the other hand, several anti-CD38 antibodies, IB4, SUN-4B7 and OKT10 (but not IB6, AT1 or AT2), induce proliferation of peripheral blood mononuclear cells (PBMC) from normal individuals (CMAusiello et al. 2000, Tissue Antigens) , 56 : 539-547).

已顯示先前技術之一些抗體能夠觸發CD38+ B細胞凋亡。然而，其僅可在基質細胞或基質衍生之細胞激素存在下執行此功能。已報導 促效性抗-CD38抗體(IB4)防止人類生髮中心(GC)B細胞凋亡(S.Zupo等人1994,Eur J Immunol,24：1218-1222)，且誘發KG-1及HL-60 AML細胞增殖(M.Konopleva等人1998,J Immunol,161：4702-4708)，但誘發Jurkat T淋巴母細胞凋亡(M.Morra等人1998,FASEB J,12：581-592)。另一抗-CD38抗體T16誘發來自ALL患者之未成熟淋巴細胞及白血病淋巴母細胞凋亡(M.Kumagai等人1995,J Exp Med,181：1101-1110)，且誘發來自AML患者之白血病骨髓母細胞凋亡(E.Todisco等人2000,Blood,95：535-542)，但T16僅在基質細胞或基質衍生之細胞激素(IL-7、IL-3、幹細胞因子)存在下誘發細胞凋亡。 Some antibodies of the prior art have been shown to be capable of triggering apoptosis of CD38 + B cells. However, it can only perform this function in the presence of stromal cells or matrix-derived cytokines. The potent anti-CD38 antibody (IB4) has been reported to prevent apoptosis in human germinal center (GC) B cells (S. Zupo et al. 1994, Eur J Immunol , 24 : 1218-1222) and induces KG-1 and HL- 60 AML cell proliferation (M. Konopleva et al. 1998, J Immunol , 161 : 4702-4708), but induced apoptosis in Jurkat T lymphoblasts (M. Morra et al. 1998, FASEB J , 12 : 581-592). Another anti-CD38 antibody T16 induces apoptosis in immature lymphocytes and leukemia lymphoblasts from ALL patients (M. Kumagai et al. 1995, J Exp Med , 181 : 1101-1110) and induces leukemia bone marrow from AML patients. Apoptosis of mother cells (E. Todisco et al. 2000, Blood , 95 : 535-542), but T16 induces cell death only in the presence of stromal cells or matrix-derived cytokines (IL-7, IL-3, stem cell factor). Die.

另一方面，一些先前技術抗體在交聯後誘發細胞凋亡，但在單獨培育時完全無任何細胞凋亡活性(WO 2006/099875)。 On the other hand, some prior art antibodies induce apoptosis after cross-linking, but are completely free of any apoptotic activity when cultured alone (WO 2006/099875).

由於CD38為用於各種造血惡性疾病之抗體療法之受關注標靶，因此吾人針對在對抗CD38-陽性惡性造血細胞之以下三種細胞毒性活性方面具高效能產生及篩選了許多種抗人類CD38抗體：誘發細胞凋亡、ADCC及CDC。本發明描述能夠經由以下三種不同細胞毒性機制殺死CD38+細胞之新穎抗-CD38抗體：誘發細胞凋亡、ADCC及CDC。顯著地，本發明最先揭示甚至在無基質細胞或基質衍生之細胞激素存在下亦能夠直接誘發CD38+細胞凋亡之抗-CD38抗體。 Since CD38 is a target of antibody therapy for various hematopoietic malignancies, we have produced and screened a wide range of anti-human CD38 antibodies against the following three cytotoxic activities against CD38-positive malignant hematopoietic cells: Apoptosis, ADCC and CDC were induced. The present invention describes novel anti-CD38 antibodies capable of killing CD38 + cells via three different cytotoxic mechanisms: induction of apoptosis, ADCC and CDC. Significantly, the present invention first discloses an anti-CD38 antibody which is capable of directly inducing apoptosis of CD38 + cells even in the absence of stromal cells or matrix-derived cytokines.

本發明之一目的在於提供特異性結合CD38且能夠經由細胞凋亡殺死CD38+細胞之抗體。一些先前技術抗體僅在交聯時能夠觸發細胞凋亡，否則無任何細胞凋亡活性，而本發明之抗體甚至在單獨培育時亦能夠誘發CD38+細胞之凋亡性細胞死亡。在本發明之一態樣中，本發明之抗體能夠經由ADCC或CDC殺死CD38+ B細胞。在另一態樣中，本發明之抗體能夠經由前述機制(亦即細胞凋亡、ADCC及CDC)中之至少兩者殺死CD38+細胞。顯著地，本發明之抗體為最先經證明 經由所有以下三種不同機制殺死CD38+ B細胞之抗-CD38抗體：細胞凋亡、ADCC及CDC。在本發明之另一實施例中，該等抗體甚至在無基質細胞或基質衍生之細胞激素存在下亦能夠經由細胞凋亡而殺死CD38+ B細胞。 It is an object of the present invention to provide an antibody that specifically binds to CD38 and is capable of killing CD38 + cells via apoptosis. Some prior art antibodies are capable of triggering apoptosis only upon cross-linking, otherwise without any apoptotic activity, whereas the antibodies of the invention are capable of inducing apoptotic cell death of CD38 + cells even when cultured alone. In one aspect of the invention, an antibody of the invention is capable of killing CD38 + B cells via ADCC or CDC. In another aspect, an antibody of the invention is capable of killing CD38 + cells via at least two of the foregoing mechanisms (ie, apoptosis, ADCC, and CDC). Significantly, the antibodies of the invention are the first anti-CD38 antibodies that have been shown to kill CD38 + B cells via all three different mechanisms: apoptosis, ADCC and CDC. In another embodiment of the invention, the antibodies are capable of killing CD38 + B cells via apoptosis even in the absence of stromal cells or matrix-derived cytokines.

本發明之抗體尤其能夠殺死惡性CD38+ B細胞，包括淋巴瘤細胞、白血病細胞及多發性骨髓瘤細胞。在一些實施例中，CD38+ B細胞為NHL、BL、MM、B-CLL、ALL、TCL、AML、HCL、HL或CML細胞。 The antibodies of the invention are particularly capable of killing malignant CD38 + B cells, including lymphoma cells, leukemia cells, and multiple myeloma cells. In some embodiments, the CD38 + B cells are NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL or CML cells.

在本發明之一態樣中，本發明之抗體能夠在無基質細胞或基質衍生之細胞激素存在下經由細胞凋亡殺死至少24%之Daudi淋巴瘤細胞及/或至少7%之Ramos淋巴瘤細胞及/或11%之MOLP-8多發性骨髓瘤細胞及/或36%之SU-DHL-8淋巴瘤細胞及/或62%之DND-41白血病細胞及/或27%之NU-DUL-1淋巴瘤細胞及/或9%之JVM-13白血病細胞及/或4%之HC-1白血病細胞。 In one aspect of the invention, an antibody of the invention is capable of killing at least 24% of Daudi lymphoma cells and/or at least 7% of Ramos lymphoma via apoptosis in the absence of stromal cells or matrix-derived cytokines Cells and / or 11% of MOLP-8 multiple myeloma cells and / or 36% of SU-DHL-8 lymphoma cells and / or 62% of DND-41 leukemia cells and / or 27% of NU-DUL- 1 lymphoma cells and / or 9% of JVM-13 leukemia cells and / or 4% of HC-1 leukemia cells.

本發明之抗體可為多株或單株抗體。較佳者為單株抗-CD38抗體。在一更佳實施例中，提供選自38SB13、38SB18、38SB19、38SB30、38SB31及38SB39之鼠科抗體，在本文中根據其輕鏈及重鏈可變區之胺基酸序列、輕鏈及重鏈可變區之基因的cDNA序列、其CDR(互補決定區)之鑑別、其表面胺基酸之鑑別及其以重組形式表現之方式對其進行全面表徵。 The antibody of the present invention may be a multi-plant or a monoclonal antibody. Preferred is a monoclonal anti-CD38 antibody. In a more preferred embodiment, a murine antibody selected from the group consisting of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 is provided herein, based on the amino acid sequence, light chain, and weight of its light and heavy chain variable regions. The cDNA sequence of the gene of the variable region of the chain, the identification of its CDR (complementarity determining region), the identification of its surface amino acid and its expression in a recombinant form are fully characterized.

本發明包括選自38SB13、38SB18、38SB19、38SB30、38SB31及38SB39之鼠科抗-CD38單株抗體之嵌合型式。亦包括38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體之表面重塑或人類化型式，其中抗體(或其抗原決定基結合片段)之輕鏈與重鏈中之可變區構架之表面暴露殘基均經置換以更接近類似於已知人類抗體表面。本發明之人類化抗體及其抗原決定基結合片段因其在所投予之人類受 檢者中具有比鼠科型式低之免疫原性(或完全無免疫原性)而具有改良之特性。因此，本發明之不同型式之人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體及其抗原決定基結合片段特異性識別CD38，同時對人類無免疫原性。 The present invention encompasses chimeric versions of murine anti-CD38 monoclonal antibodies selected from the group consisting of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39. Also included are surface remodeling or humanized versions of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies in which the surface of the variable region framework of the light chain and heavy chain of the antibody (or its epitope binding fragment) is exposed The bases are all replaced to be closer to the surface of a known human antibody. The humanized antibody of the present invention and the epitope binding fragment thereof are affected by the human being administered thereto The tester has a lower immunogenicity (or no immunogenicity) than the murine type and has improved properties. Thus, the different versions of the humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies and their epitope binding fragments of the invention specifically recognize CD38 and are not immunogenic to humans.

本發明之38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體之人類化型式在本文中根據其各別輕鏈及重鏈可變區之胺基酸序列、輕鏈及重鏈可變區之基因的DNA序列、互補決定區(CDR)之鑑別、其可變區構架表面胺基酸殘基之鑑別及其以重組形式表現之方式的揭示進行全面表徵。 The humanized versions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies of the present invention are herein based on the amino acid sequence, the light chain and heavy chain variable region genes of their respective light and heavy chain variable regions. The DNA sequence, the recognition of the complementarity determining regions (CDRs), the identification of the amino acid residues on the surface of the variable region framework, and the disclosure of the manner in which they are expressed in recombinant form are fully characterized.

本發明亦涵蓋包含(1)識別及結合CD38之細胞結合劑及(2)細胞毒性劑之細胞毒性結合物之間的結合物之用途。在細胞毒性結合物中，細胞結合劑對CD38具有高親和力且細胞毒性劑對表現CD38之細胞具高度細胞毒性，使得本發明之細胞毒性結合物形成有效殺傷劑。 The invention also encompasses the use of a combination comprising (1) a cell binding agent that recognizes and binds to CD38 and (2) a cytotoxic conjugate of a cytotoxic agent. In cytotoxic conjugates, the cell binding agent has a high affinity for CD38 and the cytotoxic agent is highly cytotoxic to cells expressing CD38, making the cytotoxic conjugate of the invention an effective killer.

在一較佳實施例中，細胞結合劑為抗-CD38抗體(例如38SB13、38SB18、38SB19、38SB30、38SB31及38SB39)或其抗原決定基結合片段，更佳為人類化抗-CD38抗體(例如38SB13、38SB18、38SB19、38SB30、38SB31及38SB39)或其抗原決定基結合片段，其中細胞毒性劑直接或經由可裂解或非可裂解連接子與抗體或其抗原決定基結合片段共價連接。在更佳實施例中，細胞結合劑為人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體或其抗原決定基結合片段，且細胞毒性劑為紫杉醇(taxol)、美登素類化合物(maytansinoid)、茅屋黴素(tomaymycin)衍生物、細黴素(leptomycin)衍生物、CC-1065或CC-1065類似物。 In a preferred embodiment, the cell binding agent is an anti-CD38 antibody (eg, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39) or an epitope binding fragment thereof, more preferably a humanized anti-CD38 antibody (eg, 38SB13) , 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39) or an epitope binding fragment thereof, wherein the cytotoxic agent is covalently linked to the antibody or its epitope binding fragment, either directly or via a cleavable or non-cleavable linker. In a more preferred embodiment, the cell binding agent is a humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibody or an epitope binding fragment thereof, and the cytotoxic agent is taxol, maytansinoid (maytansinoid) ), tomaymycin derivative, leptomycin derivative, CC-1065 or CC-1065 analog.

更佳地，細胞結合劑為人類化抗-CD38抗體38SB13、38SB18、38SB19、38SB30、38SB31及38SB39，且細胞毒性劑為美登素(maytansine)化合物，諸如DM1或DM4。 More preferably, the cell binding agent is a humanized anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39, and the cytotoxic agent is a maytansine compound such as DM1 or DM4.

本發明亦涵蓋保留結合CD38能力之抗-CD38抗體之片段的用途。在本發明之另一態樣中，涵蓋抗-CD38抗體之功能性等效物之用途。 The invention also contemplates the use of a fragment that retains a CD38-binding anti-CD38 antibody. In another aspect of the invention, the use of a functional equivalent of an anti-CD38 antibody is contemplated.

本發明亦包括一種抑制表現CD38之細胞生長之方法。在較佳實施例中，抑制表現CD38之細胞生長之方法係在活體內進行且致使細胞死亡，但亦包括活體外及離體應用。 The invention also encompasses a method of inhibiting the growth of cells expressing CD38. In a preferred embodiment, the method of inhibiting the growth of cells expressing CD38 is carried out in vivo and causes cell death, but also includes in vitro and ex vivo applications.

本發明亦提供一種包含抗-CD38抗體或抗-CD38抗體-細胞毒性劑結合物及醫藥學上可接受之載劑或賦形劑之治療組合物。在一些實施例中，治療組合物包含第二治療劑。此第二治療劑可選自包含以下各物之群：表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、肝細胞生長因子(HGF)、組織因子(TF)、蛋白質C、蛋白質S、血小板衍生之生長因子(PDGF)、神經調節素-1(heregulin)、巨噬細胞刺激蛋白(MSP)或血管內皮生長因子(VEGF)之拮抗劑；或表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、肝細胞生長因子(HGF)、組織因子(TF)、蛋白質C、蛋白質S、血小板衍生之生長因子(PDGF)、神經調節素-1、巨噬細胞刺激蛋白(MSP)或血管內皮生長因子(VEGF)之受體(包括HER2受體、HER3受體、c-MET及其他受體酪胺酸激酶)之拮抗劑。此第二治療劑亦可選自包含靶向分化群(CD)抗原之抗體之群，該等抗原包括CD3、CD14、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD36、CD40、CD44、CD52、CD55、CD59、CD56、CD70、CD79、CD80、CD103、CD134、CD137、CD138及CD152。此第二治療劑亦可選自化學治療劑或免疫調節劑之群。 The invention also provides a therapeutic composition comprising an anti-CD38 antibody or an anti-CD38 antibody-cytotoxic agent conjugate and a pharmaceutically acceptable carrier or excipient. In some embodiments, the therapeutic composition comprises a second therapeutic agent. The second therapeutic agent may be selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S , platelet-derived growth factor (PDGF), neuromodulin-1 (heregulin), macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF) antagonist; or epidermal growth factor (EGF), fibroblast Growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), neurotonin-1, macrophage stimulating protein (MSP) or An antagonist of vascular endothelial growth factor (VEGF) receptors, including the HER2 receptor, HER3 receptor, c-MET, and other receptor tyrosine kinases. The second therapeutic agent may also be selected from the group consisting of antibodies targeting a differentiation group (CD) antigen, including CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD40, CD44 , CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138 and CD152. The second therapeutic agent can also be selected from the group of chemotherapeutic agents or immunomodulators.

本發明進一步包括一種使用治療組合物治療患有癌症或發炎疾病(包括自體免疫疾病)之受檢者之方法。在一些實施例中，癌症係選自由NHL、BL、MM、B-CLL、ALL、TCL、AML、HCL、HL及CML組成之群。在另一實施例中，自體免疫疾病係選自由以下各病症組成 之群：全身性紅斑性狼瘡症、多發性硬化症、類風濕性關節炎、克隆氏病(Crohn's disease)、潰瘍性結腸炎、胃炎、橋本氏甲狀腺炎(Hashimoto's thyroiditis)、強直性脊椎炎、C型肝炎相關之冷凝球蛋白性血管炎、慢性局竈性腦炎、大疱性類天疱瘡、A型血友病、膜性增生性絲球體腎炎、修格蘭氏症候群(Sjogren's syndrome)、成人及青少年皮肌炎、成人多發性肌炎、慢性蕁麻疹、原發性膽汁性肝硬化症、特發性血小板減少性紫癜、視神經脊髓炎、格雷氏甲狀腺機能障礙疾病(Graves' dysthyroid disease)、大疱性類天疱瘡、膜性增生性絲球體腎炎、徹奇-斯全司症候群(Churg-Strauss syndrome)及哮喘。在較佳實施例中，細胞毒性結合物包含抗-CD38抗體及細胞毒性劑。在更佳實施例中，細胞毒性結合物包含人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體-DM1結合物，人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體-DM4，或人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體-紫杉烷結合物，且該結合物係連同醫藥學上可接受之載劑或賦形劑一起投予。 The invention further encompasses a method of treating a subject having cancer or an inflammatory disease, including an autoimmune disease, using a therapeutic composition. In some embodiments, the cancer is selected from the group consisting of NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL, and CML. In another embodiment, the autoimmune disease is selected from the group consisting of the following conditions Groups: systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, gastritis, Hashimoto's thyroiditis, ankylosing spondylitis, Hepatitis C-related condensed globulin vasculitis, chronic focal encephalitis, bullous pemphigoid, hemophilia A, membranous proliferative glomerulonephritis, Sjogren's syndrome, Adult and adolescent dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, optic neuromyelitis, Graves' dysthyroid disease , bullous pemphigoid, membranous proliferative spheroid nephritis, Churg-Strauss syndrome and asthma. In a preferred embodiment, the cytotoxic conjugate comprises an anti-CD38 antibody and a cytotoxic agent. In a more preferred embodiment, the cytotoxic conjugate comprises humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibody-DM1 conjugates, humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibody-DM4, or human 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibody-taxane conjugates are administered, and the combination is administered together with a pharmaceutically acceptable carrier or excipient.

在本發明之另一態樣中，使用抗-CD38抗體偵測生物樣本中之CD38蛋白。在一較佳實施例中，使用該等抗體測定腫瘤組織中之CD38含量。 In another aspect of the invention, the anti-CD38 antibody is used to detect CD38 protein in a biological sample. In a preferred embodiment, the antibodies are used to determine the amount of CD38 in tumor tissue.

本發明亦包括一種包含抗-CD38抗體或抗-CD38抗體-細胞毒性劑結合物及使用說明書之套組。在較佳實施例中，抗-CD38抗體為人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體，且細胞毒性劑為美登素化合物(諸如DM1或DM4)、茅屋黴素衍生物、細黴素衍生物或紫杉烷，且說明書係針對在治療患有癌症之受檢者中使用該等結合物。套組亦可包括製備醫藥學上可接受之調配物所必需之組份，諸如當結合物處於凍乾狀態或濃縮形式時所必需之稀釋劑，且 包括投予調配物所必需之組份。 The invention also encompasses a kit comprising an anti-CD38 antibody or an anti-CD38 antibody-cytotoxic agent conjugate and instructions for use. In a preferred embodiment, the anti-CD38 antibody is a humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibody, and the cytotoxic agent is a maytansin compound (such as DM1 or DM4), a tomaymycin derivative, and a fine A taxomycin derivative or a taxane, and the instructions are directed to the use of such conjugates in the treatment of a subject having cancer. The kit may also include components necessary for the preparation of a pharmaceutically acceptable formulation, such as those necessary when the combination is in a lyophilized or concentrated form, and This includes the components necessary to administer the formulation.

除非另有規定，否則本文中所引用之所有文獻及專利均以引用的方式併入。 All documents and patents cited herein are hereby incorporated by reference.

圖1A展示經純化之鼠科抗-CD38抗體38SB13、38SB18、38SB19與表現人類CD38之300-19細胞及CD38-陽性Ramos淋巴瘤細胞之特異性結合的FACS分析。 Figure 1A shows FACS analysis of purified murine anti-CD38 antibodies 38SB13, 38SB18, 38SB19 and specific binding of human CD38-bearing 300-19 cells and CD38-positive Ramos lymphoma cells.

圖1B展示經純化之鼠科抗-CD38抗體38SB30、38SB31、38SB39及對照抗-CD38抗體AT13/5與表現人類CD38之300-19細胞及CD38-陽性Ramos淋巴瘤細胞之特異性結合的FACS分析。 Figure 1B shows FACS analysis of purified mouse anti-CD38 antibody 38SB30, 38SB31, 38SB39 and control anti-CD38 antibody AT13/5 with specific binding to human CD38-like 300-19 cells and CD38-positive Ramos lymphoma cells. .

圖2展示以Ramos細胞確立之38SB13、38SB18、38SB19、38SB30、38SB31及38SB39之結合滴定曲線。 Figure 2 shows the binding titration curves for 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 established by Ramos cells.

圖3展示在與38SB13、38SB19或AT13/5(10nM)一起培育24h後經歷細胞凋亡之Ramos細胞之FACS點陣圖(FL4-H；TO-PRO-3染色；y軸及FL1-H；Annexin V-FITC染色；x軸)。 Figure 3 shows FACS dot plots of Ramos cells undergoing apoptosis after incubation with 38SB13, 38SB19 or AT13/5 (10 nM) for 24 h (FL4-H; TO-PRO-3 staining; y-axis and FL1-H; Annexin V-FITC staining; x-axis).

圖4A展示在與38SB13、38SB18、38SB19、38SB30、38SB31、38SB39、38SB7、38SB23、IB4、AT13/5、OKT10或SUN-4B7一起培育24h後經歷細胞凋亡之Ramos細胞之平均百分數。繪製來自雙重複樣本之Annexin V-陽性細胞之平均百分數(y軸；包括TO-PRO-3陽性與陰性細胞)。 Figure 4A shows the average percentage of Ramos cells undergoing apoptosis after incubation with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, 38SB39, 38SB7, 38SB23, IB4, AT13/5, OKT10 or SUN-4B7 for 24 h. The average percentage of Annexin V-positive cells from double replicate samples (y-axis; including TO-PRO-3 positive and negative cells) was plotted.

圖4B展示在與如圖4A相同之抗體組一起培育24h後經歷細胞凋亡之Daudi細胞之平均百分數。 Figure 4B shows the average percentage of Daudi cells undergoing apoptosis after 24 hours of incubation with the same antibody panel as Figure 4A.

圖4C展示與如圖4A相同之抗體組一起培育24h後經歷細胞凋亡之Molp-8細胞之平均百分數。 Figure 4C shows the average percentage of Molp-8 cells undergoing apoptosis after 24 hours of incubation with the same antibody panel as in Figure 4A.

圖5A展示用於表現hu38SB19-LC之人類表現載體之圖解。 Figure 5A shows an illustration of a human expression vector for expression of hu38SB19-LC.

圖5B展示用於表現hu38SB19-HC之人類表現載體之圖解。 Figure 5B shows an illustration of a human expression vector for expression of hu38SB19-HC.

圖5C展示用於表現hu38SB19LC與hu38SB19HC之人類表現載體之圖解。 Figure 5C shows an illustration of a human expression vector for expression of hu38SB19LC and hu38SB19HC.

圖6A展示對於Ramos細胞，由抗體ch38SB13、ch38SB18及ch38SB19介導之ADCC活性。 Figure 6A shows ADCC activity mediated by antibodies ch38SB13, ch38SB18 and ch38SB19 for Ramos cells.

圖6B展示對於Ramos細胞，由抗體ch38SB30、ch38SB31及ch38SB39介導之ADCC活性。 Figure 6B shows ADCC activity mediated by antibodies ch38SB30, ch38SB31 and ch38SB39 for Ramos cells.

圖7A)展示對於LP-1多發性骨髓瘤細胞，由抗體ch38SB18、ch38SB19、ch38SB31及非結合性嵌合人類IgG1對照抗體介導之ADCC活性。 Figure 7A) shows ADCC activity mediated by antibodies ch38SB18, ch38SB19, ch38SB31 and non-binding chimeric human IgGl control antibodies for LP-1 multiple myeloma cells.

圖7B)比較對於Daudi細胞由抗體ch38SB19及鼠科38SB19介導之ADCC活性。 Figure 7B) Comparison of ADCC activity mediated by antibodies ch38SB19 and murine 38SB19 for Daudi cells.

圖8A展示對於NALM-6 B-ALL細胞，由ch38SB19抗體及由非結合性嵌合人類IgG1對照抗體介導之ADCC活性。 Figure 8A shows ADCC activity mediated by the ch38SB19 antibody and by the non-binding chimeric human IgGl control antibody for NALM-6 B-ALL cells.

圖8B展示對於MOLT-4 T-ALL細胞，由ch38SB19抗體及由非結合性嵌合人類IgG1對照抗體介導之ADCC活性。 Figure 8B shows ADCC activity mediated by the ch38SB19 antibody and by the non-binding chimeric human IgGl control antibody for MOLT-4 T-ALL cells.

圖9A展示對於Raji-IMG細胞，由抗體ch38SB13、ch38SB18、ch38SB19、ch38SB30及ch38SB39介導之CDC活性。 Figure 9A shows CDC activity mediated by antibodies ch38SB13, ch38SB18, ch38SB19, ch38SB30 and ch38SB39 for Raji-IMG cells.

圖9B展示對於Raji-IMG細胞，由抗體ch38SB19及ch38SB31介導之CDC活性。 Figure 9B shows CDC activity mediated by antibodies ch38SB19 and ch38SB31 for Raji-IMG cells.

圖10展示對於LP-1多發性骨髓瘤細胞，由抗體ch38SB18、ch38SB19、ch38SB31及由非結合性嵌合人類IgG1對照抗體介導之CDC活性。 Figure 10 shows CDC activity mediated by antibodies ch38SB18, ch38SB19, ch38SB31 and by non-binding chimeric human IgGl control antibodies for LP-1 multiple myeloma cells.

圖11A展示對於Daudi細胞，由抗體ch38SB13、cch38SB19及ch38SB39介導之CDC活性。 Figure 11A shows CDC activity mediated by antibodies ch38SB13, cch38SB19 and ch38SB39 for Daudi cells.

圖11B展示對於Daudi細胞，由抗體ch38SB18及ch38SB30介導之CDC活性。 Figure 11B shows CDC activity mediated by the antibodies ch38SB18 and ch38SB30 for Daudi cells.

圖11C展示對於Daudi細胞，由抗體ch38SB19及ch38SB31介導之CDC活性。 Figure 11C shows CDC activity mediated by antibodies ch38SB19 and ch38SB31 for Daudi cells.

圖12A展示對於與Ramos細胞結合，ch38SB19、hu38SB19 v1.00及hu38SB19 v1.20之結合滴定曲線。 Figure 12A shows the binding titration curves for ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 for binding to Ramos cells.

圖12B展示針對ch38SB19、hu38SB19 v1.00及hu38SB19 v1.00與經結合生物素之鼠科38SB19抗體競爭結合Ramos細胞之能力來比較ch38SB19、hu38SB19 v1.00及hu38SB19 v1.00之結合曲線。 Figure 12B shows the binding curves for ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.00 for the ability of ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.00 to compete with the biotinylated murine 38SB19 antibody for binding to Ramos cells.

圖13展示在與ch38SB19、hu38SB19 v1.00或hu38SB19 v1.20抗體一起培育24h後經歷細胞凋亡之Daudi細胞之平均百分數。 Figure 13 shows the average percentage of Daudi cells undergoing apoptosis after 24 h incubation with ch38SB19, hu38SB19 v1.00 or hu38SB19 v1.20 antibody.

圖14展示對於LP-1多發性骨髓瘤細胞，由抗體ch38SB19、hu38SB19 v1.00、hu38SB19 v1.20及由非結合性嵌合人類IgG1對照抗體介導之ADCC活性。 Figure 14 shows ADCC activity mediated by antibodies ch38SB19, hu38SB19 v1.00, hu38SB19 v1.20 and by non-binding chimeric human IgG1 control antibodies for LP-1 multiple myeloma cells.

圖15A展示對於Raji-IMG淋巴瘤細胞，由抗體ch38SB19、hu38SB19 v1.00及hu38SB19 v1.20介導之CDC活性。 Figure 15A shows CDC activity mediated by antibodies ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 for Raji-IMG lymphoma cells.

圖15B展示對於LP-1多發性骨髓瘤細胞，由抗體ch38SB19、hu38SB19 v1.00及hu38SB19 v1.20介導之CDC活性。 Figure 15B shows CDC activity mediated by antibodies ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 for LP-1 multiple myeloma cells.

圖15C展示對於DND-41 T細胞急性淋巴母細胞白血病細胞，由抗體ch38SB19、hu38SB19 v1.00及hu38SB19 v1.20介導之CDC活性。 Figure 15C shows CDC activity mediated by antibodies ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 for DND-41 T cell acute lymphoblastic leukemia cells.

圖16展示對於SU-DHL-8彌漫性大B細胞淋巴瘤細胞、NU-DUL-1 B細胞淋巴瘤細胞、DND-41 T細胞急性淋巴母細胞白血病細胞、JVM-13 B細胞慢性淋巴細胞性白血病細胞及HC-1毛細胞白血病細胞，在與hu38SB19 v1.00抗體一起培育24h後Annexin V陽性細胞之平均百分數。 Figure 16 shows chronic lymphocyticity in SU-DHL-8 diffuse large B-cell lymphoma cells, NU-DUL-1 B-cell lymphoma cells, DND-41 T-cell acute lymphoblastic leukemia cells, and JVM-13 B cells. Leukemia cells and HC-1 hairy cell leukemia cells, the average percentage of Annexin V positive cells after 24 hours of incubation with hu38SB19 v1.00 antibody.

圖17展示負載經確立之播散性人類Ramos腫瘤之SCID小鼠之存活百分數。如圖所示，小鼠經鼠科38SB13、38SB18、38SB19、 38SB30、38SB31或38SB39抗體或PBS治療。 Figure 17 shows the percent survival of SCID mice loaded with established disseminated human Ramos tumors. As shown, the mice were subjected to the murine 38SB13, 38SB18, 38SB19, Treatment with 38SB30, 38SB31 or 38SB39 antibody or PBS.

圖18展示負載經確立之播散性人類Daudi腫瘤之SCID小鼠之存活百分數。如圖所示，小鼠經hu38SB19或mu38SB19抗體或PBS治療。 Figure 18 shows the percent survival of SCID mice loaded with established disseminated human Daudi tumors. As shown, mice were treated with hu38SB19 or mu38SB19 antibody or PBS.

圖19展示負載NCI-H929多發性骨髓瘤異種移植腫瘤之SCID小鼠之平均腫瘤體積。如圖所示，小鼠經hu38SB19、非結合性對照IgG1抗體或PBS治療。 Figure 19 shows the mean tumor volume of SCID mice bearing NCI-H929 multiple myeloma xenograft tumors. As shown, mice were treated with hu38SB19, a non-binding control IgGl antibody or PBS.

圖20展示負載MOLP-8多發性骨髓瘤異種移植腫瘤之SCID小鼠之平均腫瘤體積。如圖所示，小鼠經hu38SB19、mu38SB19、非結合性對照IgG1抗體或PBS治療。 Figure 20 shows the mean tumor volume of SCID mice bearing MOLP-8 multiple myeloma xenograft tumors. As shown, mice were treated with hu38SB19, mu38SB19, non-binding control IgG1 antibody or PBS.

本文提供能夠特異性結合CD38之新抗體。詳言之，本發明者已發現特異性結合細胞表面上之CD38且經由細胞凋亡殺死CD38+細胞之新穎抗體。在本發明之一態樣中，抗-CD38抗體亦能夠經由抗體依賴性細胞毒性(ADCC)殺死CD38+細胞。在另一態樣中，本發明之抗-CD38抗體能夠經由補體依賴性細胞毒性(CDC)殺死CD38+細胞。在另一態樣中，本發明之抗-CD38抗體能夠經由上述機制(細胞凋亡、ADCC及CDC)中之至少兩者殺死CD38+細胞。詳言之，在一較佳實施例中，本發明之抗-CD38抗體能夠經由細胞凋亡、ADCC及CDC殺死CD38+細胞。由此，本發明最先提供能夠經由三種不同機制殺死CD38+細胞之抗-CD38抗體。 Provided herein are novel antibodies that specifically bind to CD38. In particular, the inventors have discovered novel antibodies that specifically bind to CD38 on the cell surface and kill CD38 + cells via apoptosis. In one aspect of the invention, the anti-CD38 antibody is also capable of killing CD38 + cells via antibody-dependent cellular cytotoxicity (ADCC). In another aspect, the anti-CD38 antibodies of the invention are capable of killing CD38 + cells via complement dependent cytotoxicity (CDC). In another aspect, an anti-CD38 antibody of the invention is capable of killing CD38 + cells via at least two of the above mechanisms (apoptosis, ADCC and CDC). In particular, in a preferred embodiment, the anti-CD38 antibodies of the invention are capable of killing CD38 + cells via apoptosis, ADCC and CDC. Thus, the present invention is the first to provide an anti-CD38 antibody capable of killing CD38 + cells via three different mechanisms.

先前已描述能夠結合CD38且觸發CD38+細胞中之凋亡性細胞死亡之抗體(M.Kumagai等人，1995,J Exp Med,181：1101-1110；E.Todisco等人2000,Blood,95：535-542)，但本發明之抗體為最先證明在無基質細胞或基質衍生之細胞激素存在下具有細胞凋亡活性之抗體。如本文所用之術語"基質"係指腫瘤之非惡性支撐組織，其包括結締組織、血管及炎症細胞。基質細胞產生可影響癌細胞行為之生長因子及 其他物質(包括細胞激素)。如本文所用之術語"細胞激素"係指介導及調節免疫、炎症及造血之小分泌性蛋白質(例如IL-1、IL-2、IL-4、IL-5及IL-6、IFNg、IL-3、IL-7及GM-CSF)。本文中已顯示先前技術之抗體在無基質細胞或基質衍生之細胞激素存在下不能觸發凋亡性細胞死亡。與此相反，本發明之抗-CD38抗體在相同條件下顯示有效的細胞凋亡活性。 Antibodies capable of binding CD38 and triggering apoptotic cell death in CD38 + cells have been previously described (M. Kumagai et al, 1995, J Exp Med , 181 : 1101-1110; E. Todisco et al. 2000, Blood , 95 : 535-542), but the antibody of the present invention is an antibody which is first demonstrated to have apoptotic activity in the absence of stromal cells or matrix-derived cytokines. The term "matrix" as used herein refers to a non-malignant supporting tissue of a tumor, which includes connective tissue, blood vessels, and inflammatory cells. Stromal cells produce growth factors and other substances (including cytokines) that can affect the behavior of cancer cells. The term "cytokine" as used herein refers to small secreted proteins that mediate and regulate immunity, inflammation, and hematopoiesis (eg, IL-1, IL-2, IL-4, IL-5, and IL-6, IFNg, IL). -3, IL-7 and GM-CSF). It has been shown herein that prior art antibodies are unable to trigger apoptotic cell death in the absence of stromal cells or matrix-derived cytokines. In contrast, the anti-CD38 antibody of the present invention showed potent apoptotic activity under the same conditions.

在另一態樣中，本發明之抗體能夠以3×10-9M或更低之kD結合CD38蛋白。 In another aspect, the antibodies of the present invention is capable of 3 × 10 -9 M or less of CD38 k D binding protein.

如本文所用之術語"CD38"係指一種II型跨膜蛋白，其包含(例如)如Genbank寄存編號NP_001766之胺基酸序列。"CD38+細胞"為表現CD38蛋白之細胞。較佳地，CD38+細胞為哺乳動物細胞。 The term "CD38" as used herein refers to a type II transmembrane protein comprising, for example, an amino acid sequence such as Genbank Accession No. NP_001766. "CD38 + cells" are cells that express CD38 protein. Preferably, the CD38 + cells are mammalian cells.

在本發明之一實施例中，CD38+細胞為惡性細胞。在另一實施例中，CD38+細胞為B細胞。在一較佳實施例中，CD38+細胞為來源於造血惡性疾病之腫瘤細胞。在一更佳實施例中，CD38+細胞為淋巴瘤細胞、白血病細胞或多發性骨髓瘤細胞。在另一較佳實施例中，CD38+細胞為NHL、BL、MM、B-CLL、ALL、TCL、AML、HCL、HL或CML細胞。 In one embodiment of the invention, the CD38 + cells are malignant cells. In another embodiment, the CD38 + cells are B cells. In a preferred embodiment, the CD38 + cells are tumor cells derived from hematopoietic malignancies. In a more preferred embodiment, the CD38 + cells are lymphoma cells, leukemia cells or multiple myeloma cells. In another preferred embodiment, the CD38 + cells are NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL or CML cells.

因此，在一實施例中，本發明提供能夠在無基質細胞或基質衍生之細胞激素存在下殺死至少24%之Daudi淋巴瘤細胞之抗-CD38抗體。在另一實施例中，本發明之抗-CD38抗體能夠在無基質細胞或基質衍生之細胞激素存在下殺死至少7%之Ramos淋巴瘤細胞。在另一實施例中，本發明之抗-CD38抗體能夠在無基質細胞或基質衍生之細胞激素存在下殺死至少11%之MOLP-8多發性骨髓瘤細胞。在另一實施例中，本發明之抗-CD38抗體能夠在無基質細胞或基質衍生之細胞激素存在下殺死至少36%之SU-DHL-8淋巴瘤細胞。在另一實施例中，本發明之抗-CD38抗體能夠在無基質細胞或基質衍生之細胞激素存在 下殺死至少27%之NU-DUL-1淋巴瘤細胞。在另一實施例中，本發明之抗-CD38抗體能夠在無基質細胞或基質衍生之細胞激素存在下殺死至少62%之DND-41白血病細胞。在另一實施例中，本發明之抗-CD38抗體能夠在無基質細胞或基質衍生之細胞激素存在下殺死至少9%之JVM-13白血病細胞。在另一實施例中，本發明之抗-CD38抗體能夠在無基質細胞或基質衍生之細胞激素存在下殺死至少4%之HC-1白血病細胞。 Thus, in one embodiment, the invention provides an anti-CD38 antibody capable of killing at least 24% of Daudi lymphoma cells in the absence of stromal cells or matrix-derived cytokines. In another embodiment, the anti-CD38 antibodies of the invention are capable of killing at least 7% of Ramos lymphoma cells in the absence of stromal cells or matrix-derived cytokines. In another embodiment, the anti-CD38 antibodies of the invention are capable of killing at least 11% of MOLP-8 multiple myeloma cells in the absence of stromal cells or matrix-derived cytokines. In another embodiment, the anti-CD38 antibodies of the invention are capable of killing at least 36% of SU-DHL-8 lymphoma cells in the absence of stromal cells or matrix-derived cytokines. In another embodiment, the anti-CD38 antibody of the invention is capable of being present in stromal cells or matrix-derived cytokines Kill at least 27% of NU-DUL-1 lymphoma cells. In another embodiment, the anti-CD38 antibodies of the invention are capable of killing at least 62% of DND-41 leukemia cells in the absence of stromal cells or matrix derived cytokines. In another embodiment, the anti-CD38 antibodies of the invention are capable of killing at least 9% of JVM-13 leukemia cells in the absence of stromal cells or matrix derived cytokines. In another embodiment, the anti-CD38 antibodies of the invention are capable of killing at least 4% of HC-1 leukemia cells in the absence of stromal cells or matrix-derived cytokines.

抗體antibody

術語"抗體"在本文中以最廣泛含義使用且特定涵蓋任何同型(諸如IgG、IgM、IgA、IgD及IgE)之單株抗體(包括全長單株抗體)、多株抗體、多特異性抗體、嵌合抗體及抗體片段。與特異性抗原反應之抗體可藉由重組方法(諸如選擇噬菌體或類似載體中之重組抗體庫)或藉由以抗原或抗原編碼核酸免疫動物來產生。 The term "antibody" is used herein in its broadest sense and specifically encompasses any isotype (such as IgG, IgM, IgA, IgD, and IgE) monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, Chimeric antibodies and antibody fragments. Antibodies reactive with specific antigens can be produced by recombinant methods, such as selection of recombinant antibody libraries in phage or similar vectors, or by immunizing animals with antigen or antigen-encoding nucleic acids.

典型IgG抗體包含由雙硫鍵連接之兩條相同重鏈及兩條相同輕鏈。每條重鏈及輕鏈含有恆定區及可變區。各可變區含有三個稱作"互補決定區"("CDR")或"高變區"之片段，其主要負責結合抗原之抗原決定基。其通常被稱作自N末端依序編號之CDR1、CDR2及CDR3。可變區中之較高度保守部分稱作"構架區"。 A typical IgG antibody comprises two identical heavy chains and two identical light chains joined by a disulfide bond. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three fragments termed "complementarity determining regions" ("CDRs") or "hypervariable regions" which are primarily responsible for binding antigenic epitopes. It is commonly referred to as CDR1, CDR2 and CDR3 numbered sequentially from the N-terminus. The higher degree of conservation in the variable region is referred to as the "framework region."

如本文所用之"VH"或"VH"係指抗體之免疫球蛋白重鏈之可變區，包括Fv、scFv、dsFv、Fab、Fab'或F(ab')2片段之重鏈。提及"VL"或"VL"係指抗體之免疫球蛋白輕鏈之可變區，包括Fv、scFv、dsFv、Fab、Fab'或F(ab')2片段之輕鏈。 As used herein, the "V H" or "VH" refers to the variable region of the immunizing antibody immunoglobulin heavy chain, comprising two heavy chain fragments Fv, scFv, dsFv, Fab, Fab ' or F (ab'). Reference to "V L " or "VL" refers to the variable region of an immunoglobulin light chain of an antibody, including the light chain of a Fv, scFv, dsFv, Fab, Fab' or F(ab') 2 fragment.

"多株抗體"為在一或多種其他、非相同抗體之中或在此等抗體存在下產生之抗體。一般而言，多株抗體係在產生非相同抗體之數種其他B淋巴細胞存在下自一種B淋巴細胞產生。通常，多株抗體係直接自經免疫動物獲得。 A "multi-drug antibody" is an antibody produced in one or more other, non-identical antibodies or in the presence of such antibodies. In general, multiple anti-systems are produced from a B lymphocyte in the presence of several other B lymphocytes that produce non-identical antibodies. Typically, multiple strains of anti-system are obtained directly from immunized animals.

如本文所用之"單株抗體"為自大體上均質之抗體群體獲得之抗體，亦即形成此群體之抗體除可以少量存在之可能天然產生之突變體以外基本上相同。此等抗體係針對單一抗原決定基且因此具有高度特異性。 A "monoclonal antibody" as used herein is an antibody obtained from a population of substantially homogeneous antibodies, i.e., an antibody that forms such a population is substantially identical except for a naturally occurring mutant that may be present in minor amounts. These anti-systems are directed against a single epitope and are therefore highly specific.

"抗原決定基"為抗體結合之抗原位點。若抗原為聚合物(諸如蛋白質或多醣)，則抗原決定基可由相連殘基或由經抗原聚合物摺疊而緊密靠近之非相連殘基形成。在蛋白質中，由相連胺基酸形成之抗原決定基在暴露於變性溶劑後通常保留，而由非相連胺基酸形成之抗原決定基在該暴露下通常喪失。 An "antigenic determinant" is an antigenic site to which an antibody binds. If the antigen is a polymer (such as a protein or a polysaccharide), the epitope can be formed by linked residues or by non-contiguous residues that are closely bound by the antigen polymer. In proteins, the epitope formed by the linked amino acid is typically retained upon exposure to a denaturing solvent, while the epitope formed by the non-linked amino acid is typically lost upon exposure.

如本文所用之術語"KD"係指特定抗體/抗原相互作用之解離常數。 As used herein the term refers to a particular antibody solution "K D" / antigen interaction of the dissociation constant.

本發明係由新穎鼠科抗-CD38抗體(本文中之38SB13、38SB18、38SB19、38SB30、38SB31及38SB39)產生，對該等抗體根據輕鏈及重鏈之胺基酸序列、CDR之鑑別、表面胺基酸之鑑別及其以重組形式表現之方式進行全面表徵。抗體38SB13、38SB18、38SB19、38SB30、38SB31及38SB39之輕鏈及重鏈及人類化型式之一級胺基酸及DNA序列揭示於本文中。 The present invention is produced by a novel murine anti-CD38 antibody (38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 herein) which is based on the amino acid sequence of the light and heavy chains, the identification of the CDRs, and the surface. The identification of amino acids and their presentation in a recombinant form are fully characterized. The light and heavy chains of the antibodies 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 and the humanized version of the tertiary amino acid and DNA sequences are disclosed herein.

產生38SB13、38SB18、38SB19、38SB30、38SB31及38SB39鼠科抗-CD38抗體之融合瘤細胞株已分別以寄存編號PTA-7667、PTA-7669、PTA-7670、PTA-7666、PTA-7668及PTA-7671於2006年6月21日寄存於美國菌種保存中心(American Type Culture Collection)(10801 University Bld,Manassas,VA,20110-2209,USA)。 The fusion cell lines producing the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 murine anti-CD38 antibodies have been deposited under the accession numbers PTA-7667, PTA-7669, PTA-7670, PTA-7666, PTA-7668 and PTA-, respectively. 7671 was deposited on June 21, 2006 at the American Type Culture Collection (10801 University Bld, Manassas, VA, 20110-2209, USA).

本發明之範疇並不限於包含此等序列之抗體及片段。實情為特異性結合CD38且能夠經由細胞凋亡、ADCC及/或CDC殺死CD38+細胞之所有抗體及片段均在本發明之範疇內。因此，抗體及抗體片段可在其骨架、CDR、輕鏈及重鏈之胺基酸序列方面不同於抗體38SB13、 38SB18、38SB19、38SB30、38SB31及38SB39或人類化衍生物，且仍在本發明之範疇內。 The scope of the invention is not limited to antibodies and fragments comprising such sequences. It is within the scope of the invention that all antibodies and fragments that specifically bind to CD38 and are capable of killing CD38 + cells via apoptosis, ADCC and/or CDC are within the scope of the invention. Thus, antibodies and antibody fragments can differ from the antibodies 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 or humanized derivatives in the amino acid sequence of their backbone, CDR, light chain, and heavy chain, and are still in the present invention. Within the scope.

在一實施例中，本發明提供包含一或多個具有選自由SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35及36組成之群之胺基酸序列之CDR的抗體或其抗原決定基結合片段。在一較佳實施例中，本發明之抗體包含至少一條重鏈及至少一條輕鏈，且該重鏈包含三個具有選自由SEQ ID NO：1、2、3、7、8、9、13、14、15、19、20、21、25、26、27、31、32及33組成之群之胺基酸序列之連續CDR，且該輕鏈包含三個具有選自由SEQ ID NO：4、5、6、10、11、12、16、17、18、22、23、24、28、29、30、34、35及36組成之群之胺基酸序列之連續CDR。 In one embodiment, the invention provides one or more comprising having selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 Amino acid sequence of groups consisting of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36 An antibody to the CDR or an epitope binding fragment thereof. In a preferred embodiment, the antibody of the invention comprises at least one heavy chain and at least one light chain, and the heavy chain comprises three having a selected from the group consisting of SEQ ID NO: 1, 2, 3, 7, 8, 9, 13 a contiguous CDR of the amino acid sequence of the group consisting of 14, 15, 19, 20, 21, 25, 26, 27, 31, 32, and 33, and the light chain comprising three having a SEQ ID NO: 4, Successive CDRs of the amino acid sequence of the group consisting of 5, 6, 10, 11, 12, 16, 17, 18, 22, 23, 24, 28, 29, 30, 34, 35 and 36.

在一更佳實施例中，本發明之抗體包含三個具有選自SEQ ID NO：1、2、3、4、5及6之群之胺基酸序列之CDR。在另一更佳實施例中，提供一種38SB13抗體，其包含至少一條重鏈及至少一條輕鏈，且該重鏈包含具有由SEQ ID NO：1、2及3組成之胺基酸序列之三個連續CDR，且該輕鏈包含具有由SEQ ID NO：4、5及6組成之胺基酸序列之三個連續CDR。 In a more preferred embodiment, the antibody of the invention comprises three CDRs having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5 and 6. In another more preferred embodiment, a 38SB13 antibody comprising at least one heavy chain and at least one light chain, and the heavy chain comprising the amino acid sequence consisting of SEQ ID NOS: 1, 2 and 3 Contiguous CDRs, and the light chain comprises three consecutive CDRs having the amino acid sequence consisting of SEQ ID NOs: 4, 5 and 6.

在另一更佳實施例中，本發明之抗體包含三個具有選自SEQ ID NO：7、8、9、10、11及12之群之胺基酸序列之CDR。在另一更佳實施例中，提供一種38SB18抗體，其包含至少一條重鏈及至少一條輕鏈，且該重鏈包含具有由SEQ ID NO：7、8及9組成之胺基酸序列之三個連續CDR，且該輕鏈包含具有由SEQ ID NO：10、11及12組成之胺基酸序列之三個連續CDR。 In another more preferred embodiment, the antibody of the invention comprises three CDRs having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11 and 12. In another more preferred embodiment, a 38SB18 antibody comprising at least one heavy chain and at least one light chain, and the heavy chain comprising the amino acid sequence consisting of SEQ ID NOs: 7, 8 and 9 is provided Contiguous CDRs, and the light chain comprises three consecutive CDRs having the amino acid sequence consisting of SEQ ID NOs: 10, 11 and 12.

在另一更佳實施例中，本發明之抗體包含三個具有選自SEQ ID NO：13、14、15、16、17及18之群之胺基酸序列之CDR。在另一更佳 實施例中，提供一種38SB19抗體，其包含至少一條重鏈及至少一條輕鏈，且該重鏈包含具有由SEQ ID NO：13、14及15組成之胺基酸序列之三個連續CDR，且該輕鏈包含具有由SEQ ID NO：16、17及18組成之胺基酸序列之三個連續CDR。 In another more preferred embodiment, the antibody of the invention comprises three CDRs having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18. Better in another In an embodiment, a 38SB19 antibody comprising at least one heavy chain and at least one light chain, and the heavy chain comprising three consecutive CDRs having an amino acid sequence consisting of SEQ ID NOs: 13, 14 and 15 The light chain comprises three consecutive CDRs having an amino acid sequence consisting of SEQ ID NOS: 16, 17 and 18.

在另一更佳實施例中，本發明之抗體包含三個具有選自SEQ ID NO：19、20、21、22、23、24之群之胺基酸序列之CDR。在另一更佳實施例中，提供一種38SB30抗體，其包含至少一條重鏈及至少一條輕鏈，且該重鏈包含具有由SEQ ID NO：19、20及21組成之胺基酸序列之三個連續CDR，且該輕鏈包含具有由SEQ ID NO：22、23及24組成之胺基酸序列之三個連續CDR。 In another more preferred embodiment, the antibody of the invention comprises three CDRs having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, 23, 24. In another more preferred embodiment, a 38SB30 antibody comprising at least one heavy chain and at least one light chain, and the heavy chain comprising the amino acid sequence consisting of SEQ ID NOs: 19, 20 and 21 Contiguous CDRs, and the light chain comprises three consecutive CDRs having the amino acid sequence consisting of SEQ ID NOs: 22, 23 and 24.

在另一更佳實施例中，本發明之抗體包含三個具有選自SEQ ID NO：25、26、27、28、29及30之群之胺基酸序列之CDR。在另一更佳實施例中，提供一種38SB31抗體，其包含至少一條重鏈及至少一條輕鏈，且該重鏈包含具有由SEQ ID NO：25、26及27組成之胺基酸序列之三個連續CDR，且該輕鏈包含具有由SEQ ID NO：28、29及30組成之胺基酸序列之三個連續CDR。 In another more preferred embodiment, the antibody of the invention comprises three CDRs having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29 and 30. In another more preferred embodiment, a 38SB31 antibody comprising at least one heavy chain and at least one light chain, and the heavy chain comprising the amino acid sequence consisting of SEQ ID NOS: 25, 26 and 27 Contiguous CDRs, and the light chain comprises three consecutive CDRs having the amino acid sequence consisting of SEQ ID NOs: 28, 29 and 30.

在另一更佳實施例中，本發明之抗體包含三個具有選自31、32、33、34、35及36之群之胺基酸序列之CDR。在另一更佳實施例中，提供一種38SB39抗體，其包含至少一條重鏈及至少一條輕鏈，且該重鏈包含具有由SEQ ID NO：31、32及33組成之胺基酸序列之三個連續CDR，且該輕鏈包含具有由SEQ ID NO：34、35及36組成之胺基酸序列之三個連續CDR。 In another more preferred embodiment, the antibody of the invention comprises three CDRs having an amino acid sequence selected from the group consisting of 31, 32, 33, 34, 35 and 36. In another more preferred embodiment, a 38SB39 antibody comprising at least one heavy chain and at least one light chain, and the heavy chain comprising the amino acid sequence consisting of SEQ ID NOS: 31, 32 and 33 Contiguous CDRs, and the light chain comprises three consecutive CDRs having the amino acid sequence consisting of SEQ ID NOs: 34, 35 and 36.

在另一實施例中，本發明之抗-CD38抗體包含具有選自由以下各序列組成之群之胺基酸序列之VL：SEQ ID NO：38、40、42、44、46及48之VL。在一更佳實施例中，提供包含具有由SEQ ID NO：38組成之胺基酸序列之VL的38SB13抗體。在一更佳實施例中，提供包含具 有由SEQ ID NO：40組成之胺基酸序列之VL的38SB18抗體。在一更佳實施例中，提供包含具有由SEQ ID NO：42組成之胺基酸序列之VL的38SB19抗體。在一更佳實施例中，提供包含具有由SEQ ID NO：44組成之胺基酸序列之VL的38SB30抗體。在一更佳實施例中，提供包含具有由SEQ ID NO：46組成之胺基酸序列之VL的38SB31抗體。在一更佳實施例中，提供包含具有由SEQ ID NO：48組成之胺基酸序列之VL的38SB39抗體。 In another embodiment, the anti -CD38 antibody of the present invention comprises a V L having the amino acid sequence of each of the group consisting of the following sequences selected from the group consisting of: SEQ ID NO: 38,40,42,44,46 and 48 of V L. In a more preferred embodiment, a 38SB13 antibody comprising a VL having an amino acid sequence consisting of SEQ ID NO: 38 is provided. In a more preferred embodiment, a 38SB18 antibody comprising a VL having an amino acid sequence consisting of SEQ ID NO: 40 is provided. In a more preferred embodiment, a 38SB19 antibody comprising a VL having an amino acid sequence consisting of SEQ ID NO: 42 is provided. In a more preferred embodiment, a 38SB30 antibody comprising a VL having an amino acid sequence consisting of SEQ ID NO: 44 is provided. In a more preferred embodiment, a 38SB31 antibody comprising a VL having an amino acid sequence consisting of SEQ ID NO: 46 is provided. In a more preferred embodiment, a 38SB39 antibody comprising a VL having an amino acid sequence consisting of SEQ ID NO: 48 is provided.

在另一實施例中，本發明之抗體包含具有選自由SEQ ID NO：50、52、54、56、58及60組成之群之胺基酸序列之VH。在一更佳實施例中，提供包含具有由SEQ ID NO：50組成之胺基酸序列之VH的38SB13抗體。在一更佳實施例中，提供包含具有由SEQ ID NO：52組成之胺基酸序列之VH的38SB18抗體。在一更佳實施例中，提供包含具有由SEQ ID NO：54組成之胺基酸序列之VH的38SB19抗體。在一更佳實施例中，提供包含具有由SEQ ID NO：56組成之胺基酸序列之VH的38SB30抗體。在一更佳實施例中，提供包含具有由SEQ ID NO：58組成之胺基酸序列之VH的38SB31抗體。在一更佳實施例中，提供包含具有由SEQ ID NO：60組成之胺基酸序列之VH的38SB39抗體。 In another embodiment, the antibody of the invention comprises a selected from the group consisting of SEQ ID NO: 50,52,54,56,58 and 60. The amino acid sequences of the V group of H. In a more preferred embodiment, a 38SB13 antibody comprising a VH having an amino acid sequence consisting of SEQ ID NO: 50 is provided. In a more preferred embodiment, a 38SB18 antibody comprising a VH having an amino acid sequence consisting of SEQ ID NO: 52 is provided. In a more preferred embodiment, a 38SB19 antibody comprising a VH having an amino acid sequence consisting of SEQ ID NO: 54 is provided. In a more preferred embodiment, a 38SB30 antibody comprising a VH having an amino acid sequence consisting of SEQ ID NO: 56 is provided. In a more preferred embodiment, a 38SB31 antibody comprising a VH having an amino acid sequence consisting of SEQ ID NO: 58 is provided. In a more preferred embodiment, a 38SB39 antibody comprising a VH having an amino acid sequence consisting of SEQ ID NO: 60 is provided.

嵌合及人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體Chimeric and humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies

如本文所用之"嵌合抗體"為其中恆定區或其一部分經改變、置換或交換使得可變區與不同種或屬於另一抗體類別或子類之恆定區連接之抗體。"嵌合抗體"亦指其中可變區或其一部分經改變、置換或交換使得恆定區與不同種或屬於另一抗體類別或子類之可變區連接之抗體。用於產生嵌合抗體之方法在此項技術中係已知的。例如參見Morrison,1985,Science,229：1202；Oi等人，1986,BioTechniques,4：214；Gillies等人，1989,J.Immunol.Methods,125：191-202；美國專 利第5,807,715號、第4,816,567號及第4,816,397號，該等文獻之全文以引用的方式併入本文中。 A "chimeric antibody" as used herein is an antibody in which the constant region or a portion thereof is altered, substituted or exchanged such that the variable region is joined to a different species or to a constant region of another antibody class or subclass. "Chimeric antibody" also refers to an antibody in which the variable region or a portion thereof is altered, substituted or exchanged such that the constant region is joined to a different species or to a variable region of another antibody class or subclass. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science , 229 :1202; Oi et al, 1986, BioTechniques , 4:214; Gillies et al, 1989, J. Immunol. Methods , 125 : 191-202; U.S. Patent No. 5,807,715, 4,816,567 No. 4,816,397, the entire contents of each of which is incorporated herein by reference.

在本發明之一實施例中，提供38SB13、38SB18、38SB19、38SB30、38SB31及38SB39之嵌合型式。詳言之，該等嵌合型式含有至少一個人類恆定區。在一更佳實施例中，此人類恆定區為人類IgG1/κ恆定區。 In one embodiment of the invention, a mating version of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 is provided. In particular, the chimeric versions contain at least one human constant region. In a more preferred embodiment, the human constant region is a human IgG1/kappa constant region.

如本文所用之術語"人類化抗體"係指含有來源於非人類免疫球蛋白之最小序列之嵌合抗體。人類化之目的在於降低引入人類之異種抗體(諸如鼠科抗體)之免疫原性，同時保持完全抗原結合親和力及抗體特異性。人類化抗體或適於不受其他哺乳動物排斥之抗體可使用諸如表面重塑及CDR移植之數種技術來產生。如本文所用之表面重塑技術使用分子模型化、統計分析及突變之組合來改變抗體可變區之非CDR表面以類似於靶宿主之已知抗體之表面。CDR移植技術涉及將(例如)小鼠抗體之互補決定區代入人類構架域中，例如參見WO 92/22653。人類化嵌合抗體較佳具有除互補決定區(CDR)外大體上或專一地來源於相應人類抗體區之恆定區及可變區及大體上或專一地來源於非人類哺乳動物之CDR。 The term "humanized antibody" as used herein refers to a chimeric antibody comprising a minimal sequence derived from a non-human immunoglobulin. The purpose of humanization is to reduce the immunogenicity of heterologous antibodies (such as murine antibodies) introduced into humans while maintaining complete antigen binding affinity and antibody specificity. Humanized antibodies or antibodies suitable for rejection by other mammals can be produced using several techniques such as surface remodeling and CDR grafting. Surface remodeling techniques as used herein use a combination of molecular modeling, statistical analysis, and mutation to alter the non-CDR surface of an antibody variable region to resemble the surface of a known antibody of a target host. CDR grafting techniques involve the substitution of, for example, a complementarity determining region of a mouse antibody into the human framework domain, for example, see WO 92/22653. Humanized chimeric antibodies preferably have a constant region and a variable region that are substantially or exclusively derived from the corresponding human antibody region in addition to a complementarity determining region (CDR) and a CDR that is substantially or exclusively derived from a non-human mammal.

用於抗體表面重塑之策略及方法及用於降低抗體在不同宿主內之免疫原性之其他方法揭示於美國專利第5,639,641號中，該專利之全文以引用的方式併入本文中。簡言之，在一較佳方法中，(1)產生一組抗體重鏈及輕鏈可變區之位置對準以得到一組重鏈及輕鏈可變區構架表面暴露位置，其中對於所有可變區之對準位置至少約98%一致；(2)對於齧齒動物抗體(或其片段)定義一組重鏈及輕鏈可變區構架表面暴露胺基酸殘基；(3)鑑別與該組齧齒動物表面暴露胺基酸殘基最接近一致之一組重鏈及輕鏈可變區構架表面暴露胺基酸殘基；(4)除在齧齒動物抗體之互補決定區之任何殘基之任何原子之5Å內的彼等胺 基酸殘基以外，將在步驟(2)中定義之該組重鏈及輕鏈可變區構架表面暴露胺基酸殘基用在步驟(3)中鑑別之該組重鏈及輕鏈可變區構架表面暴露胺基酸殘基取代；及(5)產生具有結合特異性之人類化齧齒動物抗體。 Strategies and methods for antibody surface remodeling and other methods for reducing the immunogenicity of antibodies in different hosts are disclosed in U.S. Patent No. 5,639,641, the disclosure of which is incorporated herein in its entirety by reference. Briefly, in a preferred method, (1) the alignment of a set of antibody heavy and light chain variable regions is generated to obtain a set of heavy and light chain variable region framework surface exposed positions, wherein The aligned positions of the variable regions are at least about 98% identical; (2) for a rodent antibody (or a fragment thereof), a set of heavy chain and light chain variable region frameworks are exposed to the surface of the amino acid residues; (3) identification and The rodent surface exposed amino acid residues are closest to one of the heavy chain and light chain variable region framework surface exposed amino acid residues; (4) any residue other than the complementarity determining region of the rodent antibody Any of the amines within 5 Å of any atom In addition to the base acid residue, the group of heavy chain and light chain variable region frameworks defined in step (2) are exposed to the surface of the amino acid residue, and the group of heavy and light chains identified in step (3) can be used. The variable region framework surface is exposed to amino acid residue substitution; and (5) produces a humanized rodent antibody having binding specificity.

抗體可使用多種其他技術來人類化，該等技術包括CDR移植(EP 0 239 400、WO 91/09967、美國專利第5,530,101號及第5,585,089號)、鑲飾或表面重塑(EP 0 592 106、EP 0 519 596；Padlan E.A.,1991,Molecular Immunology 28(4/5)：489-498；Studnicka G.M.等人，1994,Protein Engineering,7(6)：805-814；Roguska M.A.等人，1994,PNAS,91：969-973)及鏈改組(美國專利第5,565,332號)。人類抗體可藉由多種此項技術中已知之方法來製成，該等方法包括噬菌體呈現法。 亦參見美國專利第4,444,887號、第4,716,111號、第5,545,806號及第5,814,318號；及國際專利申請公開案第WO 98/46645號、第WO 98/50433號、第WO 98/24893號、第WO 98/16654號、第WO 96/34096號、第WO 96/33735號及第WO 91/10741號(該等文獻之全文以引用的方式併入)。 Antibodies can be humanized using a variety of other techniques, including CDR grafting (EP 0 239 400, WO 91/09967, U.S. Patent Nos. 5,530,101 and 5,585,089), inlays or surface remodeling (EP 0 592 106, EP 0 519 596; Padlan EA, 1991, Molecular Immunology 28 (4/5) : 489-498; Studnicka GM et al, 1994, Protein Engineering , 7(6) : 805-814; Roguska MA et al, 1994, PNAS , 91 : 969-973) and chain reorganization (US Patent No. 5,565,332). Human antibodies can be made by a variety of methods known in the art, including phage display. See also U.S. Patent Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and International Patent Application Publication No. WO 98/46645, WO 98/50433, WO 98/24893, WO 98 /16654, WO 96/34096, WO 96/33735, and WO 91/10741 (the entire contents of each of which are hereby incorporated by reference).

本發明提供人類化抗體或其片段，其識別CD38且經由細胞凋亡、ADCC及/或CDC殺死CD38+細胞。在另一實施例中，人類化抗體或其抗原決定基結合片段具有經由所有三種機制殺死該等CD38+細胞之能力。在另一實施例中，本發明之人類化抗體或其抗原決定基結合片段甚至在無基質細胞或基質衍生之細胞激素存在下亦能夠經由細胞凋亡殺死該等CD38+細胞。 The invention provides humanized antibodies or fragments thereof that recognize CD38 and kill CD38 + cells via apoptosis, ADCC and/or CDC. In another embodiment, the humanized antibody or its epitope binding fragment has the ability to kill the CD38 + cells via all three mechanisms. In another embodiment, a humanized antibody or epitope-binding fragment thereof of the invention is capable of killing such CD38 + cells via apoptosis even in the absence of stromal cells or matrix-derived cytokines.

該人類化抗體之一較佳實施例為人類化38SB13、38SB18、38SB19、38SB30、38SB31或38SB39抗體或其抗原決定基結合片段。 A preferred embodiment of the humanized antibody is a humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibody or an epitope binding fragment thereof.

在更佳實施例中，提供38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體之表面重塑或人類化型式，其中抗體或其片段 之輕鏈與重鏈中之表面暴露殘基均經置換以更接近類似於已知人類抗體表面。本發明之人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體或其抗原決定基結合片段具有改良之特性。舉例而言，人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體或其抗原決定基結合片段特異性識別CD38蛋白。更佳地，人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體或其抗原決定基結合片段具有經由細胞凋亡、ADCC及/或CDC殺死CD38+細胞之額外能力。 In a more preferred embodiment, a surface remodeling or humanization version of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies is provided, wherein the light-chain and surface-exposed residues in the heavy chain of the antibody or fragment thereof are substituted Closer to the surface of a known human antibody. The humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies or epitope-binding fragments thereof of the present invention have improved properties. For example, the humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies or their epitope binding fragments specifically recognize the CD38 protein. More preferably, the humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies or epitope-binding fragments thereof have the additional ability to kill CD38 + cells via apoptosis, ADCC and/or CDC.

38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體之人類化型式在本文中亦根據其各別輕鏈及重鏈可變區之胺基酸序列、輕鏈及重鏈可變區之基因的DNA序列、CDR之鑑別、其表面胺基酸之鑑別及其以重組形式表現之方式的揭示進行全面表徵。然而，本發明之範疇並不限於包含此等序列之抗體及片段。實情為特異性結合CD38且能夠經由細胞凋亡、ADCC及/或CDC殺死CD38+細胞之所有抗體及片段均在本發明之範疇內。較佳地，該等抗體能夠經由所有三種機制殺死CD38+細胞。因此，本發明之抗體及抗原決定基結合抗體片段可在其骨架、CDR及/或輕鏈及重鏈之胺基酸序列方面不同於38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體或其人類化衍生物，且仍在本發明之範疇內。 The humanized versions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies are also herein based on the DNA of the amino acid sequence, the light chain and the heavy chain variable region of the respective light and heavy chain variable regions. The characterization of sequences, CDRs, identification of their surface amino acids, and their presentation in recombinant form is fully characterized. However, the scope of the invention is not limited to antibodies and fragments comprising such sequences. It is within the scope of the invention that all antibodies and fragments that specifically bind to CD38 and are capable of killing CD38 + cells via apoptosis, ADCC and/or CDC are within the scope of the invention. Preferably, the antibodies are capable of killing CD38 + cells via all three mechanisms. Thus, the antibody and epitope-binding antibody fragments of the invention may differ from the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies or humans thereof in terms of their backbone, CDR and/or amino acid sequences of the light and heavy chains. Derivatives are still within the scope of the invention.

38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體之CDR係藉由模型化來鑑別且已預測其分子結構。另外，儘管CDR對於抗原決定基識別具有重要性，但其並非為本發明之抗體及片段所必需。因此，提供經(例如)本發明之抗體之親和力成熟而產生之具有改良特性之抗體及片段。 The CDRs of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies were identified by modeling and their molecular structure was predicted. In addition, although CDRs are important for epitope recognition, they are not required for the antibodies and fragments of the invention. Thus, antibodies and fragments having improved properties resulting from, for example, affinity maturation of the antibodies of the invention are provided.

38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體之重鏈及輕鏈可變區之序列及其CDR之序列先前係未知的且在本申請案 中列出。該等資訊可用於產生38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體之人類化型式。此等人類化抗-CD38抗體或其衍生物亦可用作本發明之細胞結合劑。 The sequences of the heavy and light chain variable regions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies and the sequences of their CDRs were previously unknown and are in the present application Listed in. Such information can be used to generate humanized versions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies. Such humanized anti-CD38 antibodies or derivatives thereof can also be used as the cell binding agent of the present invention.

因此，在一實施例中，本發明提供包含一或多個具有選自由SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35及36組成之群之胺基酸序列之CDR的人類化抗體或其抗原決定基結合片段。在一較佳實施例中，本發明之人類化抗體包含至少一條重鏈及至少一條輕鏈，且該重鏈包含三個具有選自由SEQ ID NO：1、2、3、7、8、9、13、14、15、19、20、21、25、26、27、31、32及33組成之群之胺基酸序列之連續CDR，且該輕鏈包含三個具有選自由SEQ ID NO：4、5、6、10、11、12、16、17、18、22、23、24、28、29、30、34、35及36組成之群之胺基酸序列之連續CDR。在另一較佳實施例中，提供一種38SB13之人類化型式，其包含至少一條重鏈及至少一條輕鏈，其中該重鏈包含三個具有由SEQ ID NO：1、2及3表示之胺基酸序列之連續互補決定區，且其中該輕鏈包含三個具有由SEQ ID NO：4、5及6表示之胺基酸序列之連續互補決定區。在另一較佳實施例中，提供一種38SB18之人類化型式，其包含至少一條重鏈及至少一條輕鏈，其中該重鏈包含三個具有由SEQ ID NO：7、8及9表示之胺基酸序列之連續互補決定區，且其中該輕鏈包含三個具有由SEQ ID NO：10、11及12表示之胺基酸序列之連續互補決定區。在另一較佳實施例中，提供一種38SB19之人類化型式，其包含至少一條重鏈及至少一條輕鏈，其中該重鏈包含三個具有由SEQ ID NO：13、14及15表示之胺基酸序列之連續互補決定區，且其中該輕鏈包含三個具有由SEQ ID NO：16、17及18表示之胺基酸序列之連續互補決定區。在另一較佳實施例中，提供一種38SB30之人類 化型式，其包含至少一條重鏈及至少一條輕鏈，其中該重鏈包含三個具有由SEQ ID NO：19、20及21表示之胺基酸序列之連續互補決定區，且其中該輕鏈包含三個具有由SEQ ID NO：22、23及24表示之胺基酸序列之連續互補決定區。在另一較佳實施例中，提供一種38SB31之人類化型式，其包含至少一條重鏈及至少一條輕鏈，其中該重鏈包含三個具有由SEQ ID NO：25、26及27表示之胺基酸序列之連續互補決定區，且其中該輕鏈包含三個具有由SEQ ID NO：28、29及30表示之胺基酸序列之連續互補決定區。在另一較佳實施例中，提供一種38SB39之人類化型式，其包含至少一條重鏈及至少一條輕鏈，其中該重鏈包含三個具有由SEQ ID NO：31、32及33表示之胺基酸序列之連續互補決定區，且其中該輕鏈包含三個具有由SEQ ID NO：34、35及36表示之胺基酸序列之連續互補決定區。 Thus, in one embodiment, the invention provides one or more comprising having selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 Group of amines of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36 A humanized antibody or epitope-binding fragment thereof of a CDR of an acid sequence. In a preferred embodiment, the humanized antibody of the invention comprises at least one heavy chain and at least one light chain, and the heavy chain comprises three having a selected from the group consisting of SEQ ID NO: 1, 2, 3, 7, 8, 9 a contiguous CDR of the amino acid sequence of the group consisting of 13, 14, 15, 19, 20, 21, 25, 26, 27, 31, 32, and 33, and the light chain comprising three having a SEQ ID NO: Successive CDRs of the amino acid sequence of the group consisting of 4, 5, 6, 10, 11, 12, 16, 17, 18, 22, 23, 24, 28, 29, 30, 34, 35 and 36. In another preferred embodiment, there is provided a humanized version of 38SB13 comprising at least one heavy chain and at least one light chain, wherein the heavy chain comprises three amines having SEQ ID NOS: 1, 2 and 3 A contiguous complementarity determining region of a base acid sequence, and wherein the light chain comprises three consecutive complementarity determining regions having amino acid sequences represented by SEQ ID NOS: 4, 5 and 6. In another preferred embodiment, there is provided a humanized version of 38SB18 comprising at least one heavy chain and at least one light chain, wherein the heavy chain comprises three amines having SEQ ID NOS: 7, 8 and 9 A contiguous complementarity determining region of a base acid sequence, and wherein the light chain comprises three consecutive complementarity determining regions having amino acid sequences represented by SEQ ID NOS: 10, 11 and 12. In another preferred embodiment, there is provided a humanized version of 38SB19 comprising at least one heavy chain and at least one light chain, wherein the heavy chain comprises three amines having SEQ ID NOS: 13, 14 and 15 A contiguous complementarity determining region of a base acid sequence, and wherein the light chain comprises three consecutive complementarity determining regions having amino acid sequences represented by SEQ ID NOS: 16, 17, and 18. In another preferred embodiment, a 38SB30 human is provided a variant comprising at least one heavy chain and at least one light chain, wherein the heavy chain comprises three consecutive complementarity determining regions having amino acid sequences represented by SEQ ID NOs: 19, 20 and 21, and wherein the light chain Three consecutive complementarity determining regions having amino acid sequences represented by SEQ ID NOS: 22, 23 and 24 are included. In another preferred embodiment, there is provided a humanized version of 38SB31 comprising at least one heavy chain and at least one light chain, wherein the heavy chain comprises three amines having SEQ ID NOS: 25, 26 and 27 A contiguous complementarity determining region of a base acid sequence, and wherein the light chain comprises three consecutive complementarity determining regions having amino acid sequences represented by SEQ ID NOs: 28, 29 and 30. In another preferred embodiment, there is provided a humanized version of 38SB39 comprising at least one heavy chain and at least one light chain, wherein the heavy chain comprises three amines having SEQ ID NOS: 31, 32 and 33 A contiguous complementarity determining region of a base acid sequence, and wherein the light chain comprises three consecutive complementarity determining regions having amino acid sequences represented by SEQ ID NOs: 34, 35 and 36.

在一實施例中，本發明提供包含具有選自SEQ ID NOS：66及72之群之胺基酸序列之VH的人類化抗體或其片段。在一較佳實施例中，提供包含具有由SEQ ID NO：66表示之胺基酸序列之VH的人類化38SB19抗體。在另一較佳實施例中，提供包含具有由SEQ ID NO：72表示之胺基酸序列之VH的人類化38SB31抗體。 In one embodiment, the invention provides a humanized antibody or fragment thereof comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NOS: 66 and 72. In a preferred embodiment, a humanized 38SB19 antibody comprising a VH having the amino acid sequence represented by SEQ ID NO: 66 is provided. In another preferred embodiment, a humanized 38SB31 antibody comprising a VH having the amino acid sequence represented by SEQ ID NO: 72 is provided.

在另一實施例中，本發明提供包含具有選自SEQ ID NOS：62、64、68及70之群之胺基酸序列之VL的人類化抗體或其片段。在一較佳實施例中，提供包含具有選自SEQ ID NO：62及64之群之胺基酸序列之VL的人類化38SB19抗體。在另一較佳實施例中，提供包含具有選自SEQ ID NO：68及70之群之胺基酸序列之VL的人類化38SB31抗體。 In another embodiment, the invention provides a humanized antibody or fragment thereof comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NOS: 62, 64, 68 and 70. In a preferred embodiment, a humanized 38SB19 antibody comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 62 and 64 is provided. In another preferred embodiment, a humanized 38SB31 antibody comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 and 70 is provided.

本發明之人類化38SB19抗體及其抗原決定基結合片段亦可包括在由表1A及表1B中之灰色殘基定義之一或多個位置處之輕鏈及/或重鏈胺基酸殘基取代，該等灰色殘基表示已自原鼠科殘基變為人類抗體28E4中之相應構架表面殘基之鼠科表面構架殘基。表1B中之標星號 (*)殘基對應於人類化38SB19重鏈變異體(SEQ ID NO：65)中之鼠科回復突變。回復突變之殘基接近於CDR之殘基且係如美國專利第5,639,641號中所述或類似於在先前人類化操作中使抗原結合親和力降低之殘基的選擇進行選擇(Roguska等人，1996，美國專利申請公開案2003/0235582及2005/0118183)。 The humanized 38SB19 antibody of the present invention and its epitope binding fragment may also include light chain and/or heavy chain amino acid residues at one or more positions defined by the gray residues in Tables 1A and 1B. Substituted, these gray residues represent murine surface framework residues that have changed from the original murine residues to the corresponding framework surface residues in human antibody 28E4. The star number in Table 1B The (*) residue corresponds to the murine back mutation in the humanized 38SB19 heavy chain variant (SEQ ID NO: 65). The residue of the back mutation is close to the residue of the CDR and is selected as described in U.S. Patent No. 5,639,641 or similar to the selection of residues which have reduced antigen binding affinity in previous humanization procedures (Roguska et al., 1996, U.S. Patent Application Publication Nos. 2003/0235582 and 2005/0118183).

同樣地，本發明之人類化38SB13、38SB18、38SB30、38SB31及38SB39抗體及其抗原決定基結合片段亦可包括輕鏈及/或重鏈胺基酸殘基之取代。 Similarly, the humanized 38SB13, 38SB18, 38SB30, 38SB31 and 38SB39 antibodies of the invention and their epitope binding fragments may also include substitutions of light chain and/or heavy chain amino acid residues.

聚核苷酸、載體及宿主細胞Polynucleotides, vectors and host cells

提供編碼本發明之抗-CD38抗體之核酸。在一實施例中，核酸分子編碼抗-CD38免疫球蛋白之重鏈及/或輕鏈。在一較佳實施例中，單一核酸編碼抗-CD38免疫球蛋白之重鏈且另一核酸分子編碼抗-CD38免疫球蛋白之輕鏈。 A nucleic acid encoding an anti-CD38 antibody of the invention is provided. In one embodiment, the nucleic acid molecule encodes a heavy chain and/or a light chain of an anti-CD38 immunoglobulin. In a preferred embodiment, a single nucleic acid encodes a heavy chain of an anti-CD38 immunoglobulin and another nucleic acid molecule encodes a light chain of an anti-CD38 immunoglobulin.

在本發明之另一態樣中，提供編碼具有選自SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70及72之群之胺基酸序列之多肽的聚核苷酸。在一較佳實施例中，本發明之聚核苷酸係選自由SEQ ID NO：37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69及71組成之群。本發明並不限於該等聚核苷酸本身，而亦包括顯示與該等聚核苷酸至少80%一致之所有聚核苷酸。 In another aspect of the invention, the encoding is provided having a SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46 Polynucleotides of polypeptides of the amino acid sequence of groups of 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 and 72. In a preferred embodiment, the polynucleotide of the present invention is selected from the group consisting of SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, Groups of 65, 67, 69 and 71. The invention is not limited to the polynucleotides themselves, but also includes all polynucleotides that are at least 80% identical to the polynucleotides.

本發明提供包含本發明之聚核苷酸之載體。在一實施例中，載體含有編碼抗-CD38免疫球蛋白之重鏈之聚核苷酸。在另一實施例中，該聚核苷酸編碼抗-CD38免疫球蛋白之輕鏈。本發明亦提供包含編碼融合蛋白、經修飾抗體、抗體片段及其探針之聚核苷酸分子之載 體。 The invention provides vectors comprising the polynucleotides of the invention. In one embodiment, the vector contains a polynucleotide encoding a heavy chain of an anti-CD38 immunoglobulin. In another embodiment, the polynucleotide encodes a light chain of an anti-CD38 immunoglobulin. The invention also provides a polynucleotide molecule comprising a fusion protein, a modified antibody, an antibody fragment and a probe thereof. body.

為表現本發明之抗-CD38抗體之重鏈及/或輕鏈，將編碼該等重鏈及/或輕鏈之聚核苷酸插入表現載體中使得基因可操作地連接至轉錄及轉譯序列。表現載體包括質體、YAC、黏質體、反轉錄病毒、EBV衍生之離合染色小體，及熟習此項技術者已知有利於確保該等重鏈及/或輕鏈表現之所有其他載體。熟習此項技術者應瞭解可將編碼重鏈及輕鏈之聚核苷酸選殖至不同載體中或同一載體中。在一較佳實施例中，將該等聚核苷酸選殖於同一載體中。 To represent the heavy and/or light chain of an anti-CD38 antibody of the invention, a polynucleotide encoding the heavy and/or light chain is inserted into an expression vector such that the gene is operably linked to the transcriptional and translational sequences. Expression vectors include plastids, YACs, vesicles, retroviruses, EBV-derived clutch-stained chromosomes, and all other vectors known to those skilled in the art to help ensure the performance of such heavy and/or light chains. Those skilled in the art will recognize that polynucleotides encoding heavy and light chains can be cloned into different vectors or in the same vector. In a preferred embodiment, the polynucleotides are selected in the same vector.

本發明之聚核苷酸及包含此等分子之載體可用於轉化合適之哺乳動物宿主細胞。轉化可藉由用於將聚核苷酸引入細胞宿主中之任何已知方法來達成。該等方法為熟習此項技術者所熟知且包括葡聚糖介導之轉化、磷酸鈣沈澱、聚凝胺介導之轉染、原生質體融合、電穿孔、將聚核苷酸封裝入脂質體中、將DNA基因槍注射及直接微注射至細胞核中。 The polynucleotides of the invention and vectors comprising such molecules can be used to transform a suitable mammalian host cell. Transformation can be achieved by any known method for introducing a polynucleotide into a cellular host. Such methods are well known to those skilled in the art and include dextran mediated transformation, calcium phosphate precipitation, polyglycol mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides into liposomes In the middle, DNA gene gun injection and direct microinjection into the nucleus.

抗體片段Antibody fragment

本發明之抗體包括上文所論述之全長抗體以及其抗原決定基結合片段。如本文所用之"抗體片段"包括保留與由全長抗體識別之抗原決定基結合之能力的抗體之任何部分，一般稱作"抗原決定基結合片段"。抗體片段之實例包括(但不限於)Fab、Fab'及F(ab')2、Fd、單鏈Fv(scFv)、單鏈抗體、雙硫鍵連接之Fv(dsFv)及包含VL或VH區之片段。抗原決定基結合片段(包括單鏈抗體)可包含單獨或與以下各者之整體或一部分組合之可變區：鉸鏈區、CH1、CH2及CH3域。 Antibodies of the invention include the full length antibodies discussed above as well as epitope binding fragments thereof. An "antibody fragment" as used herein includes any portion of an antibody that retains the ability to bind to an epitope recognized by a full length antibody, commonly referred to as an "antigenic determinant binding fragment." Examples of antibody fragments include, but are not limited to, Fab, Fab' and F(ab') 2 , Fd, single chain Fv (scFv), single chain antibodies, disulfide linked Fv (dsFv), and VL or VH containing regions Fragment. An epitope binding fragment (including a single chain antibody) can comprise a variable region, either alone or in combination with one or more of the following: the hinge region, the CH1, CH2, and CH3 domains.

此等片段可含有一或兩個Fab片段或F(ab')2片段。儘管含有少於完整抗體之所有六個CDR(諸如三個、四個或五個CDR)之片段亦具有功能性，但較佳地抗體片段含有所有該等區。此外，片段可為或可組合以下免疫球蛋白類別中之任一者之成員：IgG、IgM、IgA、IgD或 IgE及其子類。 Such fragments may contain one or two Fab fragments or F(ab') 2 fragments. While fragments containing less than all six CDRs of an intact antibody (such as three, four or five CDRs) are also functional, it is preferred that the antibody fragments contain all of such regions. Furthermore, a fragment can be or can be a member of any of the following immunoglobulin classes: IgG, IgM, IgA, IgD or IgE and subclasses thereof.

Fab及F(ab')2片段可藉由蛋白水解裂解，使用諸如木瓜蛋白酶(Fab片段)或胃蛋白酶(F(ab')2片段)之酶來產生。 Fab and F(ab') 2 fragments can be produced by proteolytic cleavage using an enzyme such as papain (Fab fragment) or pepsin (F(ab') 2 fragment).

"單鏈Fv"("scFvs")片段為含有與至少一個抗體輕鏈可變區(VL)片段連接之至少一個抗體重鏈可變區(VH)片段之抗原決定基結合片段。連接子可為短的可撓性肽，該肽經選擇以確保在VL區與VH區連接後發生該等區之正確三維摺疊以維持單鏈抗體片段來源之完整抗體之靶分子結合特異性。VL或VH序列之羧基末端可經由連接子與互補VL或VH序列之胺基酸末端共價連接。 "Single-chain Fv" ( "scFvs") fragments are containing at least one antibody heavy chain variable region (V H) connected to the at least one antibody light chain variable region (V L) fragment of the epitope binding fragment thereof. The linker can be a short flexible peptide which is selected to ensure that the correct three-dimensional folding of the regions occurs after attachment of the V L region to the V H region to maintain the specific binding of the target antibody to the intact antibody from which the single-chain antibody fragment is derived. Sex. Carboxyl terminus of V L or V H sequence of amino acids may be coupled via a linker to a complementary V H or V L sequences covalently terminal.

本發明之單鏈抗體片段含有具有本說明書中所述之完整抗體之可變區或互補決定區(CDR)中之至少一者，但缺乏彼等抗體之一些或所有恆定域的胺基酸序列。此等恆定域並非抗原結合所必需，但構成完整抗體結構之主要部分。單鏈抗體片段可由此克服與使用含有一部分或所有恆定域之抗體相關之一些問題。舉例而言，單鏈抗體片段傾向於無生物分子與重鏈恆定區之間的不當相互作用，或無其他不合需要之生物活性。另外，單鏈抗體片段比完整抗體小得多且可由此具有大於完整抗體之毛細管滲透性，從而使單鏈抗體片段可更有效地定位及結合靶抗原結合位點。另外，抗體片段可在原核細胞中以相對較大規模產生，由此有利於其產生。此外，單鏈抗體片段之相對較小尺寸使其比完整抗體在接受者中引起免疫反應之可能性較小。 A single-chain antibody fragment of the invention comprises an amino acid sequence having at least one of a variable region or a complementarity determining region (CDR) of an intact antibody as described herein, but lacking some or all of the constant domains of the antibodies . These constant domains are not required for antigen binding, but constitute a major part of the intact antibody structure. Single-chain antibody fragments can thereby overcome some of the problems associated with the use of antibodies containing some or all of the constant domains. For example, single-chain antibody fragments tend to have no undue interaction between the biomolecule and the heavy chain constant region, or no other undesirable biological activity. In addition, single-chain antibody fragments are much smaller than intact antibodies and can thus have greater capillary permeability than intact antibodies, thereby allowing single-chain antibody fragments to more efficiently localize and bind to target antigen binding sites. In addition, antibody fragments can be produced on a relatively large scale in prokaryotic cells, thereby facilitating their production. Furthermore, the relatively small size of a single-chain antibody fragment makes it less likely to cause an immune response in the recipient than the intact antibody.

單鏈抗體片段可藉由分子選殖、抗體噬菌體呈現庫或熟習此項技術者所熟知之類似技術來產生。此等蛋白質可(例如)在真核細胞或原核細胞(包括細菌)中產生。本發明之抗原決定基結合片段亦可使用此項技術中已知之各種噬菌體呈現方法來產生。在噬菌體呈現方法中，在載有編碼功能性抗體域之聚核苷酸序列之噬菌體粒子之表面上呈現功能性抗體域。詳言之，可使用該噬菌體呈現自全套或組合抗體 庫(例如人類或鼠科)所表現之抗原決定基結合域。表現與所關注抗原結合之抗原決定基結合域之噬菌體可用抗原(例如使用結合或俘獲於固體表面或珠粒之經標記抗原)來選擇或鑑別。此等方法中所用之噬菌體通常為包括自具有與噬菌體基因III或基因VIII蛋白重組融合之Fab、Fv或雙硫鍵穩定之Fv抗體域之噬菌體所表現之fd及M13結合域的絲狀噬菌體。 Single-chain antibody fragments can be produced by molecular selection, antibody phage display libraries, or similar techniques well known to those skilled in the art. Such proteins can be produced, for example, in eukaryotic cells or prokaryotic cells, including bacteria. The epitope binding fragments of the invention can also be produced using a variety of phage display methods known in the art. In the phage display method, a functional antibody domain is presented on the surface of a phage particle carrying a polynucleotide sequence encoding a functional antibody domain. In particular, the phage can be presented from a full or combined antibody The epitope binding domain represented by a library (eg, human or murine). Phage displaying a epitope binding domain that binds to the antigen of interest can be selected or identified using an antigen (e.g., using a labeled antigen that binds or is captured on a solid surface or bead). The phage used in these methods are usually filamentous phage including fd and M13 binding domains represented by phage having Fab, Fv or a disulfide-stabilized Fv antibody domain fused recombinantly with phage gene III or gene VIII protein.

可用於產生本發明之抗原決定基結合片段之噬菌體呈現方法之實例包括以下文獻中所揭示之方法：Brinkman等人，1995,J.Immunol.Methods,182：41-50；Ames等人，1995,J.Immunol.Methods,184：177-186；Kettleborough等人，1994,Eur.J.Immunol.,24：952-958；Persic等人，1997,Gene,187：9-18；Burton等人，1994,Advances in Immunology,57：191-280；WO/1992/001047、WO 90/02809、WO 91/10737、WO 92/01047、WO 92/18619、WO 93/11236、WO 95/15982、WO 95/20401；及美國專利第5,698,426號、第5,223,409號、第5,403,484號、第5,580,717號、第5,427,908號、第5,750,753號、第5,821,047號、第5,571,698號、第5,427,908號、第5,516,637號、第5,780,225號、第5,658,727號、第5,733,743號及第5,969,108號；該等文獻之各者之全文均以引用的方式併入本文中。 Examples of phage display methods that can be used to generate the epitope binding fragments of the invention include those disclosed in Brinkman et al, 1995, J. Immunol. Methods , 182 : 41-50; Ames et al, 1995, J. Immunol. Methods , 184 : 177-186; Kettleborough et al, 1994, Eur . J. Immunol ., 24 : 952-958; Persic et al, 1997, Gene , 187 : 9-18; Burton et al., 1994 , Advances in Immunology , 57 : 191-280; WO/1992/001047, WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/ U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5, 658, 727, 5, 733, 743, and 5, 969, 108; the entire contents of each of which are incorporated herein by reference.

在噬菌體選擇後，(例如)如下文詳細描述，可將編碼片段之噬菌體區域分離且用其使用重組DNA技術經由在包括哺乳動物細胞、昆蟲細胞、植物細胞、酵母及細菌之所選宿主中表現來產生抗原決定基結合片段。舉例而言，用以重組產生Fab、Fab'及F(ab')2片段之技術亦可使用諸如以下文獻中所揭示之方法的此項技術中已知之方法來使用，WO 92/22324；Mullinax等人，1992,BioTechniques,12(6)：864-869；Sawai等人，1995,AJRI,34：26-34；及Better等人，1988,Science, 240：1041-1043；該等文獻之全文以引用的方式併入。可用於產生單鏈Fv及抗體之技術之實例包括以下文獻中所述之技術：美國專利第4,946,778號及第5,258,498號；Huston等人，1991,Methods in Enzymology,203：46-88；Shu等人，1993,PNAS,90：7995-7999；Skerra等人，1988,Science,240：1038-1040。 Following phage selection, for example, as described in detail below, the phage region encoding the fragment can be isolated and used to express in a selected host including mammalian cells, insect cells, plant cells, yeast, and bacteria using recombinant DNA techniques. To generate an epitope binding fragment. For example, techniques for recombinant production of Fab, Fab' and F(ab') 2 fragments can also be used using methods known in the art, such as those disclosed in the literature, WO 92/22324; Mullinax Et al., 1992, BioTechniques , 12(6) : 864-869; Sawai et al, 1995, AJRI , 34 : 26-34; and Better et al, 1988, Science , 240 : 1041-1043; Incorporated by reference. Examples of techniques that can be used to generate single-chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology , 203 :46-88; Shu et al. , 1993, PNAS , 90 :7995-7999; Skerra et al., 1988, Science , 240 : 1038-1040.

功能性等效物Functional equivalent

本發明之範疇內亦包括抗-CD38抗體及人類化抗-CD38受體抗體之功能性等效物。術語"功能性等效物"包括具有同源序列之抗體、嵌合抗體、人工抗體及經修飾抗體，例如其中各功能性等效物係由其與CD38蛋白結合之能力來定義。熟習此項技術者應瞭解稱作"抗體片段"之分子之群與稱作"功能性等效物"之群存在重疊。產生功能性等效物之方法為熟習此項技術者所已知且揭示於(例如)WO 93/21319、EP 239,400、WO 89/09622、EP 338,745及EP 332,424中，該等文獻之全文分別以引用的方式併入。 Functional equivalents of anti-CD38 antibodies and humanized anti-CD38 receptor antibodies are also included within the scope of the invention. The term "functional equivalent" includes antibodies, chimeric antibodies, artificial antibodies, and modified antibodies having homologous sequences, for example, wherein each functional equivalent is defined by its ability to bind to a CD38 protein. Those skilled in the art will recognize that there is an overlap between a population of molecules called "antibody fragments" and a group called "functional equivalents." Methods of producing functional equivalents are known to those skilled in the art and are disclosed, for example, in WO 93/21319, EP 239,400, WO 89/09622, EP 338,745, and EP 332,424, the entire contents of each of which are The manner of reference is incorporated.

具有同源序列之抗體為具有與本發明之抗-CD38抗體及人類化抗-CD38抗體之胺基酸序列具有序列同源性之胺基酸序列的彼等抗體。較佳地，同源性為與本發明之抗-CD38抗體及人類化抗-CD38抗體之可變區之胺基酸序列的同源性。如本文中應用於胺基酸序列之"序列同源性"定義為如(例如)根據Pearson及Lipman,1988,Proc.Natl.Acad.Sci.USA,85：2444-2448藉由FASTA搜索法所測定，與另一胺基酸序列具有至少約90%、91%、92%、93%或94%序列同源性，且更佳至少約95%、96%、97%、98%或99%序列同源性之序列。 An antibody having a homologous sequence is an antibody having an amino acid sequence having sequence homology to the amino acid sequence of the anti-CD38 antibody of the present invention and the humanized anti-CD38 antibody. Preferably, the homology is homology to the amino acid sequence of the variable regions of the anti-CD38 antibody and the humanized anti-CD38 antibody of the invention. "Sequence homology" as applied herein to an amino acid sequence is defined, for example, by the FASTA search method according to Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA , 85 : 2444-2448. Determined to have at least about 90%, 91%, 92%, 93% or 94% sequence homology to another amino acid sequence, and more preferably at least about 95%, 96%, 97%, 98% or 99% Sequence of sequence homology.

人工抗體包括scFv片段、微型雙功能抗體、微型三功能抗體、微型四功能抗體及mru(參見Winter,G.及Milstein,C.,1991,Nature,349：293-299；Hudson,P.J.,1999,Current Opinion in Immunology,11：548-557之論述)，該等人工抗體之各者均具有抗原結合能力。在單鏈Fv片 段(scFv)中，抗體之VH及VL域係經由可撓性肽連接。通常，此連接肽長約15個胺基酸殘基。若連接子更小，例如5個胺基酸，則形成微型雙功能抗體，其為二價scFv二聚體。若連接子減至少於三個胺基酸殘基，則形成被稱作微型三功能抗體及微型四功能抗體之三聚及四聚結構。抗體之最小結合單位為CDR，通常為具有充分特異性識別及結合而可單獨使用之重鏈CDR2。此片段稱作分子識別單位或mru。數個此等mru可經短連接肽連接在一起，由此形成具有高於單一mru之親和力之人工結合蛋白。 Artificial antibodies include scFv fragments, mini-bifunctional antibodies, mini-trifunctional antibodies, mini-four-function antibodies, and mru (see Winter, G. and Milstein, C., 1991, Nature , 349 : 293-299; Hudson, PJ, 1999, Current Opinion in Immunology , 11 : 548-557), each of these artificial antibodies has antigen binding ability. In a single-chain Fv fragment (scFv), the VH and VL domains of the antibody are linked via a flexible peptide. Typically, this linker peptide is about 15 amino acid residues long. If the linker is smaller, such as five amino acids, a minibifunctional antibody is formed which is a divalent scFv dimer. If the linker is reduced by at least three amino acid residues, a trimeric and tetrameric structure called a minitrifunctional antibody and a minitetrafunctional antibody is formed. The minimal binding unit of an antibody is a CDR, typically a heavy chain CDR2 that can be used alone with sufficient specific recognition and binding. This fragment is called a molecular recognition unit or mru. Several of these mru can be joined together via short linker peptides, thereby forming an artificial binding protein with an affinity greater than that of a single mru.

本申請案之功能性等效物亦包括經修飾抗體，例如藉由任何類型之分子與抗體共價連接而經修飾之抗體。舉例而言，經修飾抗體包括已(例如)藉由糖基化、乙醯化、聚乙二醇化、磷酸化、醯胺化、由已知保護基/阻隔基衍生化、蛋白水解裂解、與細胞配位子或其他蛋白質連接等而經修飾之抗體。共價連接不阻礙抗體產生抗獨特型反應。此等修飾可藉由已知技術來進行，該等技術包括(但不限於)特定化學裂解、乙醯化、甲醯化、衣黴素之代謝合成等。另外，經修飾抗體可含有一或多種非常規胺基酸。 Functional equivalents of the present application also include modified antibodies, such as antibodies modified by covalent attachment of any type of molecule to an antibody. For example, modified antibodies include, for example, by glycosylation, acetylation, pegylation, phosphorylation, guanidine, derivatization from known protecting groups/barriers, proteolytic cleavage, and An antibody modified by a cell ligand or other protein linkage. Covalent attachment does not prevent the antibody from producing an anti-idiotype response. Such modifications can be made by known techniques including, but not limited to, specific chemical cleavage, acetylation, formazanization, metabolic synthesis of tunicamycin, and the like. Additionally, the modified antibody may contain one or more unconventional amino acids.

功能性等效物可藉由互換不同構架內不同鏈上之不同CDR來產生。因此，舉例而言，藉由取代不同重鏈，針對一組特定CDR可產生不同類別之抗體，藉此可產生(例如)IgG1-4、IgM、IgA1-2、IgD、IgE抗體類型及同型。類似地，本發明之範疇內之人工抗體可藉由將一組特定CDR嵌入完全合成構架內來產生。 Functional equivalents can be produced by exchanging different CDRs on different chains within different frameworks. Thus, for example, by substituting different heavy chains, different classes of antibodies can be produced for a particular set of CDRs, thereby producing, for example, IgGl, IgM, IgA1-2, IgD, IgE antibody types and isotypes. Similarly, artificial antibodies within the scope of the present invention can be produced by embedding a specific set of CDRs into a fully synthetic framework.

功能性等效物可易於藉由使用此項技術中已知之多種方法在側接一組特定CDR之可變區及/或恆定區序列內執行突變、缺失及/或插入來產生。本發明之抗體片段及功能性等效物涵蓋當與38SB13、38SB18、38SB19、38SB30、38SB31或38SB39抗體相比時與CD38具有可偵側結合程度之彼等分子。可偵側結合程度包括在鼠科 38SB13、38SB18、38SB19、38SB30、38SB31或38SB39抗體與CD38之結合能力之至少10-100%，較佳至少50%、60%或70%，更佳至少75%、80%、85%、90%、95%或99%之範圍內的所有值。 Functional equivalents can be readily generated by performing mutations, deletions, and/or insertions within the variable region and/or constant region sequences flanking a particular set of CDRs using a variety of methods known in the art. Antibody fragments and functional equivalents of the invention encompass those molecules that have detectable side binding to CD38 when compared to 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibodies. The degree of detectable side binding is included in the murine At least 10-100%, preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90% of the binding capacity of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibody to CD38 , all values in the range of 95% or 99%.

經改良抗體Modified antibody

CDR對於抗原決定基識別及抗體結合具有首要重要性。然而，可對構成CDR之殘基作出改變而不干擾抗體識別及結合其同源抗原決定基之能力。舉例而言，可作出不影響抗原決定基識別，而增加抗體對抗原決定基之結合親和力之改變。 CDRs are of primary importance for epitope recognition and antibody binding. However, alterations can be made to the residues that make up the CDRs without interfering with the ability of the antibody to recognize and bind to its cognate epitope. For example, changes can be made that do not affect epitope recognition, but increase the binding affinity of the antibody to the epitope.

因此，本發明之範疇內亦包括鼠科與人類化抗體之經改良型式，其亦特異性識別及結合CD38，並較佳具有增加之親和力。 Accordingly, a modified version of murine and humanized antibodies is also included within the scope of the invention which also specifically recognizes and binds to CD38 and preferably has increased affinity.

數項研究已基於一級抗體序列之瞭解來研究在抗體序列中之多個位置處引入一或多種胺基酸改變對其特性(諸如結合及表現量)之影響(Yang,W.P.等人，1995,J.Mol.Biol.,254：392-403；Rader,C.等人，1998,Proc.Natl.Acad.Sci.USA,95：8910-8915；Vaughan,T.J.等人，1998,Nature Biotechnology,16：535-539)。 Several studies have investigated the effect of introducing one or more amino acids at various positions in an antibody sequence on its properties, such as binding and amount of expression, based on the knowledge of primary antibody sequences (Yang, WP et al, 1995, J. Mol. Biol. , 254 : 392-403; Rader, C. et al., 1998, Proc. Natl. Acad. Sci. USA , 95 : 8910-8915; Vaughan, TJ et al., 1998, Nature Biotechnology , 16 :535-539).

在此等研究中，藉由使用諸如寡核苷酸介導之定點誘變、盒式誘變、易錯PCR、DNA改組或大腸桿菌(E.coli)之突變菌株之方法改變CDR1、CDR2、CDR3或構架區中之重鏈及輕鏈基因之序列來產生一級抗體之等效物(Vaughan,T.J.等人，1998,Nature Biotechnology,16：535-539；Adey,N.B.等人，1996，第16章，第277-291頁，於"Phage Display of Peptides and Proteins"中，編者Kay,B.K.等人，Academic Press)。改變一級抗體之序列之此等方法使得二級抗體之親和力得以改良(Gram,H.等人，1992,Proc.Natl.Acad.Sci.USA,89：3576-3580；Boder,E.T.等人，2000,Proc.Natl.Acad.Sci.USA,97：10701-10705；Davies,J.及Riechmann,L.,1996,Immunotechnolgy,2：169-179；Thompson,J.等人，1996,J.Mol.Biol.,256：77-88；Short, M.K.等人，2002,J.Biol.Chem.,277：16365-16370；Furukawa,K.等人，2001,J.Biol.Chem.,276：27622-27628)。 In these studies, CDR1, CDR2 were altered by methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutant strains of E. coli . Sequences of heavy and light chain genes in CDR3 or framework regions to produce equivalents of primary antibodies (Vaughan, TJ et al, 1998, Nature Biotechnology , 16 : 535-539; Adey, NB et al, 1996, 16th Chapters, pp. 277-291, in "Phage Display of Peptides and Proteins", editor Kay, BK et al., Academic Press). Such methods of altering the sequence of the primary antibody result in improved affinity of the secondary antibody (Gram, H. et al., 1992, Proc. Natl. Acad. Sci. USA , 89 : 3576-3580; Boder, ET et al., 2000). , Proc. Natl. Acad. Sci. USA , 97 : 10701-10705; Davies, J. and Riechmann, L., 1996, Immunotechnolgy , 2 : 169-179; Thompson, J. et al., 1996, J. Mol. Biol ., 256 :77-88; Short, MK et al., 2002, J. Biol . Chem ., 277 : 16365-16370; Furukawa, K. et al., 2001, J. Biol . Chem ., 276 :27622- 27628).

藉由改變抗體之一或多個胺基酸殘基之類似定向策略，可使用本發明中所述之抗體序列研發具有經改良功能(包括針對CD38之經改良親和力)之抗-CD38抗體。 An anti-CD38 antibody having improved functionality, including improved affinity for CD38, can be developed using the antibody sequences described herein by altering the targeting strategy of one or more amino acid residues of the antibody.

較佳之胺基酸取代為：(1)可降低蛋白質水解之敏感性，(2)可降低氧化之敏感性，(3)可改變形成蛋白質複合物之結合親和力，及(4)可賦予或修飾該等類似物之其他物理化學或功能特性之胺基酸取代。 類似物可包括序列不同於天然產生之肽序列之各種突變蛋白質。舉例而言，可在天然產生之序列中，較佳在多肽形成分子間接觸之域以外之部分中進行單或多胺基酸取代(較佳為保守胺基酸取代)。保守胺基酸取代不應實質上改變親本序列之結構特徵(例如，置換胺基酸不應傾向於斷裂存在於親本序列中之螺旋，或破壞表徵親本序列之其他類型二級結構)。此項技術認知之多肽二級結構及三級結構之實例描述於Proteins,Structures and Molecular Principles(Creighton編，W.H.Freeman and Company,New York(1984))；Introduction to Protein Structure(C.Branden及J.Tooze編，Garland Publishing,New York,N.Y.(1991))；及Thornton等人，1991,Nature,354：105中，該等文獻各併入本文供參考。 Preferred amino acid substitutions are: (1) reduced sensitivity to proteolysis, (2) reduced oxidative sensitivity, (3) altered binding affinity for protein complex formation, and (4) imparted or modified Amino acid substitutions of other physicochemical or functional properties of such analogs. Analogs can include a variety of mutant proteins that differ in sequence from naturally occurring peptide sequences. For example, a mono- or polyamino acid substitution (preferably a conservative amino acid substitution) can be carried out in a naturally occurring sequence, preferably in a portion other than the domain in which the polypeptide is formed into an intermolecular contact. Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (eg, the replacement amino acid should not tend to cleave the helix present in the parent sequence, or destroy other types of secondary structures that characterize the parent sequence) . Examples of polypeptide secondary and tertiary structures of this technology are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984); Introduction to Protein Structure (C. Branden and J. Tooze). Ed., Garland Publishing, New York, NY (1991); and Thornton et al, 1991, Nature , 354 :105, each of which is incorporated herein by reference.

改良抗體亦包括該等藉由動物免疫、融合瘤形成及選擇具有特異性特徵之抗體的標準技術所製備之具有改良特徵之抗體。 Modified antibodies also include such improved antibodies prepared by standard techniques of animal immunization, fusion neoplasia, and selection of antibodies with specific characteristics.

本發明之改良抗體尤其包括具有增強之功能特性之抗體。尤其受關注者為該等具有增強介導細胞毒性效應功能(諸如ADCC)之能力之抗體。該等抗體可藉由在抗體之恆定構架中進行單或多取代以改變其與Fc受體之相互作用來獲得。用於設計此等突變體之方法可見於例如Lazar等人(2006,Proc.Natl.Acad.Sci.U.S.A. 103(11)：4005-4010)及 Okazaki等人(2004,J.Mol.Biol. 336(5)：1239-49)中。亦參見WO 03/074679、WO 2004/029207、WO 2004/099249、WO2006/047350、WO 2006/019447、WO 2006/105338、WO 2007/041635。亦可使用經特定工程以產生改良抗體之細胞株。詳言之，此等細胞株已改變糖基化路徑之調節，產生極少岩藻糖基化或甚至完全去岩藻糖基化之抗體。該等細胞株及其工程方法揭示於例如Shinkawa等人(2003,J.Biol.Chem. 278(5)：3466-3473),Ferrara等人(2006,J.Biol.Chem. 281(8)：5032-5036；2006,Biotechnol.Bioeng. 93(5)：851-61),EP 1331266、EP 1498490、EP 1498491、EP 1676910、EP 1792987及WO 99/54342中。 Improved antibodies of the invention include, inter alia, antibodies having enhanced functional properties. Of particular interest are those antibodies that have the ability to enhance the ability to mediate cytotoxic effects, such as ADCC. Such antibodies can be obtained by single or multiple substitutions in the constant framework of the antibody to alter its interaction with the Fc receptor. Methods for designing such mutants can be found, for example, in Lazar et al. (2006, Proc. Natl. Acad. Sci. USA 103 (11): 4005-4010) and Okazaki et al. (2004, J. Mol. Biol. 336 (5): 1239-49). See also WO 03/074679, WO 2004/029207, WO 2004/099249, WO 2006/047350, WO 2006/019447, WO 2006/105338, WO 2007/041635. Cell lines that have been engineered to produce improved antibodies can also be used. In particular, these cell lines have altered the regulation of the glycosylation pathway, resulting in antibodies that are rarely fucosylated or even completely defucosylated. Such cell lines and engineering methods thereof are disclosed, for example, in Shinkawa et al. (2003, J. Biol. Chem. 278 (5): 3466-3473), Ferrara et al. (2006, J. Biol. Chem. 281 (8): . 5032-5036; 2006, Biotechnol.Bioeng 93 ( 5): 851-61), EP 1331266, EP 1498490, EP 1498491, EP 1676910, EP 1792987 and in WO 99/54342.

本發明亦包括細胞毒性結合物。此等細胞毒性結合物包含兩種主要組份，細胞結合劑及細胞毒性劑。 The invention also includes cytotoxic conjugates. These cytotoxic conjugates comprise two major components, a cell binding agent and a cytotoxic agent.

如本文所用之術語"細胞結合劑"係指特異性識別及結合細胞表面上之CD38蛋白之藥劑。在一實施例中，細胞結合劑特異性識別CD38使其允許結合物以靶向方式發揮作用而具有極少由非特異性結合所產生之副作用。 The term "cell binding agent" as used herein refers to an agent that specifically recognizes and binds to CD38 protein on the surface of a cell. In one embodiment, the cell binding agent specifically recognizes CD38 such that it allows the conjugate to function in a targeted manner with minimal side effects resulting from non-specific binding.

在另一實施例中，本發明之細胞結合劑亦特異性識別CD38蛋白使得結合物可與靶細胞接觸足夠時間段以允許結合物之細胞毒性藥物部分作用於細胞及/或給予結合物由細胞內在化之足夠時間。 In another embodiment, the cell binding agent of the invention also specifically recognizes the CD38 protein such that the conjugate can be contacted with the target cell for a sufficient period of time to allow the cytotoxic drug moiety of the conjugate to act on the cell and/or to administer the conjugate by the cell Internal time is sufficient.

在一較佳實施例中，細胞毒性結合物包含抗-CD38抗體作為細胞結合劑，更佳包含鼠科38SB13、38SB18、38SB19、38SB30、38SB31或38SB39抗-CD38單株抗體。在另一較佳實施例中，細胞結合劑為該抗-CD38抗體之嵌合型式。在一更佳實施例中，細胞毒性結合物包含人類化38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體或其抗原決定基結合片段。38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體能夠特異性識別CD38，且以靶向方式將細胞毒性劑定向於異常細胞或組織(諸如癌細胞)。 In a preferred embodiment, the cytotoxic conjugate comprises an anti-CD38 antibody as a cell binding agent, more preferably a murine 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 anti-CD38 monoclonal antibody. In another preferred embodiment, the cell binding agent is a chimeric version of the anti-CD38 antibody. In a more preferred embodiment, the cytotoxic conjugate comprises a humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibody or an epitope binding fragment thereof. The 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies are capable of specifically recognizing CD38 and targeting cytotoxic agents to abnormal cells or tissues, such as cancer cells, in a targeted manner.

本發明之細胞毒性結合物之第二組份為細胞毒性劑。如本文所用之術語"細胞毒性劑"係指降低或阻斷細胞之功能或生長及/或引起細胞破壞之物質。 The second component of the cytotoxic conjugate of the invention is a cytotoxic agent. The term "cytotoxic agent" as used herein refers to a substance that reduces or blocks the function or growth of a cell and/or causes cell destruction.

在較佳實施例中，細胞毒性劑為小分子藥物、前藥、紫杉類藥物(taxoid)、美登素類化合物(諸如DM1或DM4)、茅屋黴素衍生物、細黴素衍生物、CC-1065或CC-1065類似物。在較佳實施例中，本發明之細胞結合劑係直接或經由可裂解或非可裂解連接子與細胞毒性劑共價連接。 In a preferred embodiment, the cytotoxic agent is a small molecule drug, a prodrug, a taxoid, a maytansinoid compound (such as DM1 or DM4), a tomaymycin derivative, a leptomycin derivative, CC-1065 or CC-1065 analog. In a preferred embodiment, the cell binding agent of the invention is covalently linked to a cytotoxic agent either directly or via a cleavable or non-cleavable linker.

細胞結合劑、細胞毒性劑及連接子在下文中更詳細地論述。 Cell binding agents, cytotoxic agents, and linkers are discussed in more detail below.

細胞結合劑Cell binding agent

本發明之化合物作為治療劑之效用視謹慎選擇適當細胞結合劑而定。細胞結合劑可為目前已知或成為已知之任何種類，且包括肽及非肽。細胞結合劑可為可以特異性或非特異性方式結合細胞之任何化合物。一般而言，此等化合物可為抗體(尤其是單株抗體)、淋巴因子、激素、生長因子、維生素、營養轉運分子(諸如轉鐵蛋白)或任何其他細胞結合分子或物質。 The utility of the compounds of the invention as therapeutic agents will depend on the careful selection of the appropriate cell binding agent. The cell binding agent can be of any class currently known or known, and includes peptides and non-peptides. The cell binding agent can be any compound that can bind to cells in a specific or non-specific manner. In general, such compounds can be antibodies (especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient transporters (such as transferrin) or any other cell binding molecule or substance.

可使用之細胞結合劑之更特定實例包括：a)多株抗體；b)單株抗體；c)諸如Fab、Fab'及F(ab')2、Fv之抗體片段(Parham,1983,J.Immunol.,131：2895-2902；Spring等人，1974,J.Immunol.,113：470-478；Nisonoff等人，1960,Arch.Biochem.Biophys.,89：230-244)；詳言之，選自38SB13、38SB18、38SB19、38SB30、38SB31及38SB39之抗-CD38單株抗體可用作本發明之細胞結合劑。同樣地，該細胞結合劑可為38SB13、38SB18、38SB19、38SB30、38SB31及 38SB39單株抗體之一之嵌合型式。較佳地，使用人類化抗-CD38抗體作為本發明之細胞結合劑。更佳地，人類化抗-CD38抗體係選自人類化或表面重塑38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體。 More specific examples of cell binding agents that can be used include: a) multiple antibodies; b) monoclonal antibodies; c) antibody fragments such as Fab, Fab' and F(ab') 2 , Fv (Parham, 1983, J. Immunol ., 131 : 2895-2902; Spring et al., 1974, J. Immunol ., 113 : 470-478; Nisonoff et al., 1960, Arch . Biochem . Biophys ., 89 : 230-244); An anti-CD38 monoclonal antibody selected from the group consisting of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 can be used as the cell binding agent of the present invention. Similarly, the cell binding agent can be a chimeric version of one of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 monoclonal antibodies. Preferably, a humanized anti-CD38 antibody is used as the cell binding agent of the present invention. More preferably, the humanized anti-CD38 anti-system is selected from the group consisting of humanized or surface remodeled 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies.

細胞毒性劑Cytotoxic agent

在另一實施例中，人類化抗體或其抗原決定基結合片段可與藥物(諸如美登素類化合物)結合形成藉由將藥物靶向CD38蛋白而對抗原表現細胞具有特異性細胞毒性之前藥。包含該等抗體及小分子高毒性藥物(例如美登素類化合物、紫杉烷、茅屋黴素衍生物、細黴素衍生物及CC-1065類似物)之細胞毒性結合物可用作用於治療諸如淋巴瘤、白血病及多發性骨髓瘤之腫瘤的治療劑。 In another embodiment, the humanized antibody or epitope-binding fragment thereof can be combined with a drug (such as a maytansinoid) to form a drug that is specifically cytotoxic to the antigen-presenting cell by targeting the drug to the CD38 protein. . Cytotoxic conjugates comprising such antibodies and small molecule highly toxic drugs (eg, maytansinoids, taxanes, tomaymycin derivatives, leptomycin derivatives, and CC-1065 analogs) can be used for treatments such as A therapeutic agent for lymphoma, leukemia, and tumors of multiple myeloma.

本發明之細胞毒性結合物中所用之細胞毒性劑可為致使細胞死亡或誘發細胞死亡或以某種方式降低細胞生存力之任何化合物。較佳之細胞毒性劑包括(例如)下文所定義之美登素類化合物及美登素類化合物類似物、紫杉類藥物、茅屋黴素衍生物、細黴素衍生物、CC-1065及CC-1065類似物、海兔毒素及海兔毒素類似物。此等細胞毒性劑與如本文所揭示之抗體、抗體片段、功能性等效物、經改良抗體及其類似物結合。 The cytotoxic agent used in the cytotoxic conjugate of the present invention may be any compound which causes cell death or induces cell death or somehow reduces cell viability. Preferred cytotoxic agents include, for example, the maytansinoids and maytansinoid analogs, taxanes, tomaymycin derivatives, leptomycin derivatives, CC-1065 and CC-1065, as defined below. Analogs, dolastatin and dolastatin analogues. Such cytotoxic agents are combined with antibodies, antibody fragments, functional equivalents, modified antibodies and analogs thereof as disclosed herein.

細胞毒性結合物可藉由活體外方法來製備。為使藥物或前藥與抗體連接，使用連接基團。合適之連接基團在此項技術中係熟知的且包括雙硫基、硫醚基、酸不穩定基團、光不穩定基團、肽酶不穩定基團及酯酶不穩定基團。較佳之連接基團為雙硫基及硫醚基。舉例而言，結合物可使用雙硫鍵交換反應或藉由在抗體與藥物或前藥之間形成硫醚鍵來建構。 Cytotoxic conjugates can be prepared by in vitro methods. In order to link the drug or prodrug to the antibody, a linking group is used. Suitable linking groups are well known in the art and include bisthio, thioether, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Preferred linking groups are dithio and thioether groups. For example, the conjugate can be constructed using a disulfide exchange reaction or by forming a thioether bond between the antibody and the drug or prodrug.

美登素類化合物Maytansinoids

可在本發明中使用以形成細胞毒性結合物之細胞毒性劑包括美 登素類化合物及美登素類化合物類似物。合適之美登素類化合物之實例包括美登醇及美登醇類似物。美登素類化合物為抑制微管形成且對哺乳動物細胞具有高度毒性之藥物。 Cytotoxic agents that can be used in the present invention to form cytotoxic conjugates include Derivatives and maytansinoid analogs. Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Maytansinoids are drugs that inhibit microtubule formation and are highly toxic to mammalian cells.

合適之美登醇類似物之實例包括具有經修飾芳環之美登醇類似物及在其他位置處具有修飾之美登醇類似物。該等合適之美登素類化合物揭示於美國專利第4,424,219號、第4,256,746號、第4,294,757號、第4,307,016號、第4,313,946號、第4,315,929號、第4,331,598號、第4,361,650號、第4,362,663號、第4,364,866號、第4,450,254號、第4,322,348號、第4,371,533號、第6,333,410號、第5,475,092號、第5,585,499號及第5,846,545號中。 Examples of suitable maytansinoid analogs include maytansinoid analogs having a modified aromatic ring and maytansinoid analogs having modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219, 4,256,746, 4,294,757, 4,307,016, 4,313,946, 4,315,929, 4,331,598, 4,361,650, 4,362,663, 4,364,866. No. 4,450,254, 4,322,348, 4,371,533, 6,333,410, 5,475,092, 5,585,499, and 5,846,545.

具有經修飾芳環之美登醇之合適類似物之特定實例包括：(1)C-19-去氯(美國專利第4,256,746號)(藉由安沙托辛P2(ansamytocin P2)之LAH還原來製備)；(2)C-20-羥基(或C-20-去甲基)+/-C-19-去氯(美國專利第4,361,650號及第4,307,016號)(藉由使用鏈黴菌(Streptomyces)或放線菌(Actinomyces)去甲基或使用LAH去氯來製備)；及(3)C-20-去甲氧基、C-20-醯氧基(-OCOR)+/-去氯(美國專利第4,294,757號)(藉由使用醯基氯進行醯化來製備)。 Specific examples of suitable analogs of the dexamethasone having a modified aromatic ring include: (1) C-19-dechlorination (U.S. Patent No. 4,256,746) (prepared by LAH reduction of ansamtotocin P2) (2) C-20-hydroxy (or C-20-demethyl) +/- C-19-dechlorinated (U.S. Patent Nos. 4,361,650 and 4,307,016) (by using Streptomyces or pay-off Actinomyces demethylation or LAH dechlorination); and (3) C-20-demethoxy, C-20-decyloxy (-OCOR) +/- dechlorination (US Patent 4,294,757) No.) (prepared by deuteration using hydrazine chloride).

具有其他位置修飾之美登醇之合適類似物之特定實例包括：(1)C-9-SH(美國專利第4,424,219號)(藉由使美登醇與H2S或P2S5反應來製備)；(2)C-14-烷氧基甲基(去甲氧基/CH2OR)(美國專利第4,331,598號)；(3)C-14-羥甲基或醯氧基甲基(CH2OH或CH2OAc)(美國專利第4,450,254號)(自土壤絲菌屬(Nocardia)製備)；(4)C-15-羥基/醯氧基(美國專利第4,364,866號)(藉由以鏈黴菌轉 化美登醇來製備)；(5)C-15-甲氧基(美國專利第4,313,946號及第4,315,929號)(自滑桃樹(Trewia nudiflora)分離)；(6)C-18-N-去甲基(美國專利第4,362,663號及第4,322,348號)(藉由以鏈黴菌使美登醇去甲基化來製備)；及(7)4,5-去氧(美國專利第4,371,533號)(藉由美登醇之三氯化鈦/LAH還原來製備)。 Specific examples of suitable analogs of maytansinol having other positional modifications include: (1) C-9-SH (U.S. Patent No. 4,424,219) (prepared by reacting maytansinol with H2S or P2S5); (2) C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Patent No. 4,331,598); (3) C-14-hydroxymethyl or decyloxymethyl (CH2OH or CH2OAc) (US Patent No. 4,450,254) (prepared from the genus Nocardia); (4) C-15-hydroxy/decyloxy (U.S. Patent No. 4,364,866) (by Streptomyces) (5) C-15-methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929) (Separated from Trewia nudiflora); (6) C-18-N- Demethylation (U.S. Patent Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces); and (7) 4,5-deoxygenation (U.S. Patent No. 4,371,533) Prepared by reduction of titanium trichloride/LAH of maytansinol).

在一較佳實施例中，本發明之細胞毒性結合物利用含硫醇之美登素類化合物(DM1)，形式上稱作N2'-去乙醯基-N2'-(3-巰基-1-側氧基丙基)-美登素，作為細胞毒性劑。DM1由以下結構式(I)表示： In a preferred embodiment, the cytotoxic conjugate of the present invention utilizes a thiol-containing maytansinoid compound (DM1), which is hereinafter referred to as N2'-desethyl-N2'-(3-mercapto-1- Sideoxypropyl)-maytansine, as a cytotoxic agent. DM1 is represented by the following structural formula (I):

在另一較佳實施例中，本發明之細胞毒性結合物利用含硫醇之美登素類化合物N2'-去乙醯基-N-2'(4-甲基-4-巰基-1-側氧基戊基)-美登素作為細胞毒性劑。DM4由以下結構式(II)表示： In another preferred embodiment, the cytotoxic conjugate of the present invention utilizes a thiol-containing maytansinoid compound N2'-desethyl-N-2' (4-methyl-4-mercapto-1-one side Oxypentyl)-maytansine acts as a cytotoxic agent. DM4 is represented by the following structural formula (II):

在本發明之其他實施例中，可使用其他美登素，包括在帶有硫原子之碳原子上帶有單烷基或二烷基取代之含硫醇及含雙硫鍵之美登素類化合物。此等化合物包括在C-3、C-14羥甲基、C-15羥基或C-20去甲基處具有含帶有受阻氫硫基之醯基之醯化胺基酸側鏈的美登素類化合物，其中帶有硫醇官能基之醯基之碳原子具有一或兩個取代基，該等取代基為CH3、C2H5、具有1至10個碳原子之直鏈或支鏈烷基或烯基、具有3至10個碳原子之環烷基或環烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基，且此外其中該等取代基之一可為H，且其中醯基在羰基官能基與硫原子之間具有至少三個碳原子之直鏈鏈長。 In other embodiments of the invention, other maytansin may be used, including thiol-containing and disulfide-containing maytansinoids having a monoalkyl or dialkyl substitution on a carbon atom bearing a sulfur atom. . These compounds include the presence of a deuterated amino acid side chain having a mercapto group having a hindered hydrogenthio group at a C-3, C-14 hydroxymethyl group, a C-15 hydroxy group or a C-20 demethyl group. a compound of the formula wherein the carbon atom having a mercapto group having a thiol functional group has one or two substituents, which are CH 3 , C 2 H 5 , a straight chain or a branch having 1 to 10 carbon atoms. An alkyl or alkenyl group, a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocycloalkyl group, and further wherein such substitution One of the groups may be H, and wherein the fluorenyl group has a linear chain length of at least three carbon atoms between the carbonyl functional group and the sulfur atom.

該等其他美登素包括由式(III)表示之化合物： 其中：Y'表示：(CR7R8)l(CR9=CR10)p(C≡C)qAr(CR5R6)mDu(CR11=CR12)r(C≡C)sBt(CR3R4)nCR1R2SZ，其中：R1及R2各自獨立地為CH3、C2H5、具有1至10個碳原子之直鏈烷 基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基，且此外R2可為H；A、B、D為具有3-10個碳原子之環烷基或環烯基、簡單或經取代之芳基或雜環芳族基團或雜環烷基；R3、R4、R5、R6、R7、R8、R9、R11及R12各自獨立地為H、CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基；l、m、n、o、p、q、r、s及t各自獨立地為0或1至5之整數，其限制條件為l、m、n、o、p、q、r、s及t中之至少兩者在任何時候不均為零；且Z為H、SR或-COR，其中R為具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基，或簡單或經取代之芳基，或雜環芳族基團或雜環烷基。 These other maytansin include compounds represented by formula (III): Where: Y' means: (CR 7 R 8 ) l (CR 9 =CR 10 ) p (C≡C) q A r (CR 5 R 6 ) m D u (CR 11 =CR 12 ) r (C≡C s B t (CR 3 R 4 ) n CR 1 R 2 SZ, wherein: R 1 and R 2 are each independently CH 3 , C 2 H 5 , a linear alkyl group having 1 to 10 carbon atoms or an alkene a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocycloalkyl group, and further R 2 may be H; A, B, D are a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, a simple or substituted aryl or heterocyclic aromatic group or a heterocycloalkyl group; R 3 , R 4 , R 5 And R 6 , R 7 , R 8 , R 9 , R 11 and R 12 are each independently H, CH 3 , C 2 H 5 , a linear alkyl or alkenyl group having 1 to 10 carbon atoms, having 3 a branched or cyclic alkyl or alkenyl group of 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocycloalkyl group; l, m, n, o, p, q, r, s, and t are each independently 0 or an integer from 1 to 5, with the constraint that at least two of l, m, n, o, p, q, r, s, and t are not zero at any time. And Z is H, SR or -COR, which R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl group, or a heterocyclic aromatic group Family or heterocycloalkyl.

式(III)之較佳實施例包括以下式(III)化合物，其中：R1為H，R2為甲基且Z為H。 Preferred embodiments of formula (III) include the following compounds of formula (III) wherein: R 1 is H, R 2 is methyl and Z is H.

R1及R2為甲基且Z為H。 R 1 and R 2 are methyl groups and Z is H.

R1為H，R2為甲基，且Z為-SCH3。 R 1 is H, R 2 is a methyl group, and Z is -SCH 3 .

R1及R2為甲基，且Z為-SCH3。 R 1 and R 2 are a methyl group, and Z is -SCH 3 .

該等其他美登素亦包括由式(IV-L)、(IV-D)或(IV-D,L)表示之化合物： 其中：Y表示(CR7R8)l(CR5R6)m(CR3R4)nCR1R2SZ，其中：R1及R2各自獨立地為CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基，且此外R2可為H；R3、R4、R5、R6、R7及R8各自獨立地為H、CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基；l、m及n各自獨立地為1至5之整數，且此外n可為0；Z為H、SR或-COR，其中R為具有1至10個碳原子之直鏈或支鏈烷基或烯基、具有3至10個碳原子之環烷基或環烯基，或簡單或經取代之芳基，或雜環芳族基團或雜環烷基；且May表示在C-3、C-14羥甲基、C-15羥基或C-20去甲基處帶有側鏈之美登素類化合物。 These other maytansines also include compounds represented by formula (IV-L), (IV-D) or (IV-D, L): Wherein: Y represents (CR 7 R 8 ) l (CR 5 R 6 ) m (CR 3 R 4 ) n CR 1 R 2 SZ, wherein: R 1 and R 2 are each independently CH 3 , C 2 H 5 , a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aromatic group Or a heterocycloalkyl group, and further R 2 may be H; R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently H, CH 3 , C 2 H 5 , having 1 to 10 a linear alkyl or alkenyl group of a carbon atom, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocycloalkyl group ;l, m and n are each independently an integer from 1 to 5, and further n may be 0; Z is H, SR or -COR, wherein R is a linear or branched alkyl group having 1 to 10 carbon atoms Or an alkenyl group, a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl group, or a heterocyclic aromatic group or a heterocycloalkyl group; and May is represented at C-3, A maytansinoid compound having a side chain at the C-14 hydroxymethyl group, a C-15 hydroxy group or a C-20 demethyl group.

式(IV-L)、(IV-D)及(IV-D,L)之較佳實施例包括以下式(IV-L)、(IV-D)及(IV-D,L)之化合物，其中：R1為H，R2為甲基，R5、R6、R7及R8各自為H，l及m各自為1，n為0，且Z為H。 Preferred examples of the formulae (IV-L), (IV-D) and (IV-D, L) include compounds of the following formulae (IV-L), (IV-D) and (IV-D, L), Wherein R 1 is H, R 2 is a methyl group, R 5 , R 6 , R 7 and R 8 are each H, 1 and m are each 1, n is 0, and Z is H.

R1及R2為甲基，R5、R6、R7、R8各自為H，l及m為1，n為0，且Z為H。 R 1 and R 2 are methyl groups, R 5 , R 6 , R 7 and R 8 are each H, l and m are 1, n is 0, and Z is H.

R1為H，R2為甲基，R5、R6、R7、R8各自為H，l及m各自為1，n為0，且Z為-SCH3。 R 1 is H, R 2 is a methyl group, R 5 , R 6 , R 7 and R 8 are each H, 1 and m are each 1, n is 0, and Z is -SCH 3 .

R1及R2為甲基，R5、R6、R7、R8各自為H，l及m為1，n為0，且Z為-SCH3。 R 1 and R 2 are a methyl group, R 5 , R 6 , R 7 and R 8 are each H, l and m are 1, n is 0, and Z is -SCH 3 .

較佳地，細胞毒性劑由式(IV-L)表示。 Preferably, the cytotoxic agent is represented by the formula (IV-L).

該等其他美登素亦包括由式(V)表示之化合物： 其中：Y表示(CR7R8)l(CR5R6)m(CR3R4)nCR1R2SZ，其中：R1及R2各自獨立地為CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基，且此外R2可為H；R3、R4、R5、R6、R7及R8各自獨立地為H、CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基；l、m及n各自獨立地為1至5之整數，且此外n可為0；且Z為H、SR或-COR，其中R為具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基，或簡單或經取代之芳基，或雜環芳族基團或雜環烷基。 These other maytansines also include compounds represented by formula (V): Wherein: Y represents (CR 7 R 8 ) l (CR 5 R 6 ) m (CR 3 R 4 ) n CR 1 R 2 SZ, wherein: R 1 and R 2 are each independently CH 3 , C 2 H 5 , a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aromatic group Or a heterocycloalkyl group, and further R 2 may be H; R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently H, CH 3 , C 2 H 5 , having 1 to 10 a linear alkyl or alkenyl group of a carbon atom, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocycloalkyl group ;l, m and n are each independently an integer from 1 to 5, and further n may be 0; and Z is H, SR or -COR, wherein R is a linear alkyl group or alkene having 1 to 10 carbon atoms a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl group, or a heterocyclic aromatic group or a heterocycloalkyl group.

式(V)之較佳實施例包括以下式(V)化合物，其中：R1為H，R2為甲基，R5、R6、R7及R8各自為H；l及m各自為1；n 為0；且Z為H。 Preferred embodiments of formula (V) include the following compounds of formula (V) wherein: R 1 is H, R 2 is methyl, R 5 , R 6 , R 7 and R 8 are each H; and l and m are each 1; n is 0; and Z is H.

R1及R2為甲基，R5、R6、R7及R8各自為H，l及m為1；n為0；且Z為H。 R 1 and R 2 are methyl groups, R 5 , R 6 , R 7 and R 8 are each H, and l and m are 1; n is 0; and Z is H.

R1為H，R2為甲基，R5、R6、R7及R8各自為H，l及m各自為1，n為0，且Z為-SCH3。 R 1 is H, R 2 is a methyl group, R 5 , R 6 , R 7 and R 8 are each H, 1 and m are each 1, n is 0, and Z is -SCH 3 .

R1及R2為甲基，R5、R6、R7及R8各自為H，l及m為1，n為0，且Z為-SCH3。 R 1 and R 2 are methyl groups, R 5 , R 6 , R 7 and R 8 are each H, l and m are 1, n is 0, and Z is -SCH 3 .

該等其他美登素進一步包括由式(VI-L)、(VI-D)或(VI-D,L)表示之化合物： 其中：Y2表示(CR7R8)l(CR5R6)m(CR3R4)nCR1R2SZ2，其中：R1及R2各自獨立地為CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基，且此外R2可為H；R3、R4、R5、R6、R7及R8各自獨立地為H、CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基；l、m及n各自獨立地為1至5之整數，且此外n可為0；Z2為SR或-COR，其中R為具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基，或簡單或經取代 之芳基，或雜環芳族基團或雜環烷基；且May為美登素類化合物。 These other maytansines further include compounds represented by formula (VI-L), (VI-D) or (VI-D, L): Wherein Y 2 represents (CR 7 R 8 ) l (CR 5 R 6 ) m (CR 3 R 4 ) n CR 1 R 2 SZ 2 , wherein: R 1 and R 2 are each independently CH 3 , C 2 H 5. A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aromatic group a group or a heterocycloalkyl group, and further R 2 may be H; R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently H, CH 3 , C 2 H 5 , having 1 to a linear alkyl or alkenyl group of 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocyclic ring Alkyl; l, m and n are each independently an integer from 1 to 5, and further n may be 0; Z 2 is SR or -COR, wherein R is a linear alkyl group or alkene having 1 to 10 carbon atoms a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl group, or a heterocyclic aromatic group or a heterocycloalkyl group; and May is a maytansinoid Compound.

該等其他美登素亦包括由式(VII)表示之化合物： 其中：Y2'表示(CR7R8)l(CR9=CR10)p(C≡C)qAr(CR5R6)mDu(CR11=CR12)r(C≡C)sBt(CR3R4)nCR1R2SZ2，其中：R1及R2各自獨立地為CH3、C2H5、具有1至10個碳原子之直鏈或支鏈烷基或烯基、具有3至10個碳原子之環烷基或環烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基，且此外R2可為H；A、B及D各自獨立地為具有3至10個碳原子之環烷基或環烯基、簡單或經取代之芳基或雜環芳族基團或雜環烷基；R3、R4、R5、R6、R7、R8、R9、R11及R12各自獨立地為H、CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基； l、m、n、o、p、q、r、s及t各自獨立地為0或1至5之整數，其限制條件為l、m、n、o、p、q、r、s及t中之至少兩者在任何時候不均為零；且Z2為SR或-COR，其中R為具有1至10個碳原子之直鏈烷基或烯基、具有3-10個碳原子之支鏈或環狀烷基或烯基，或簡單或經取代之芳基，或雜環芳族基團或雜環烷基。 These other maytansines also include compounds represented by formula (VII): Where: Y 2 ' represents (CR 7 R 8 ) l (CR 9 =CR 10 ) p (C≡C) q A r (CR 5 R 6 ) m D u (CR 11 =CR 12 ) r (C≡C s B t (CR 3 R 4 ) n CR 1 R 2 SZ 2 , wherein: R 1 and R 2 are each independently CH 3 , C 2 H 5 , a straight or branched chain having 1 to 10 carbon atoms An alkyl or alkenyl group, a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocycloalkyl group, and further R 2 may be H ; A, B and D are each independently a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, a simple or substituted aryl or heterocyclic aromatic group or a heterocycloalkyl group; R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 11 and R 12 are each independently H, CH 3 , C 2 H 5 , a linear alkyl group having 1 to 10 carbon atoms or an alkene a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocycloalkyl group; l, m, n, o, p, q, r, s, and t are each independently 0 or an integer from 1 to 5, with the constraint being at least two of l, m, n, o, p, q, r, s, and t at any time Unevenness is zero; and Z 2 Is SR or -COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or simply or substituted An aryl group, or a heterocyclic aromatic group or a heterocycloalkyl group.

式(VII)之較佳實施例包括以下式(VII)化合物，其中：R1為H且R2為甲基。 Preferred embodiments of formula (VII) include the following compounds of formula (VII) wherein: R 1 is H and R 2 is methyl.

上述美登素類化合物可與抗-CD38抗體38SB13、38SB18、38SB19、38SB30、38SB31或38SB39或其同源物或片段結合，其中該抗體係使用存在於美登素類化合物之C-3、C-14羥甲基、C-15羥基或C-20去甲基處所見之醯化胺基酸側鏈之醯基上的硫醇或雙硫鍵官能基與美登素類化合物連接，且其中醯化胺基酸側鏈之醯基之硫醇或雙硫鍵官能基位於具有一或兩個取代基之碳原子處，該等取代基為CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基，且此外該等取代基之一可為H，且其中醯基在羰基官能基與硫原子之間具有至少三個碳原子之直鏈鏈長。 The above maytansinoid compound can be combined with an anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 or a homologue or fragment thereof, wherein the anti-system uses C-3, C present in the maytansinoid compound a thiol or disulfide bond on the thiol group of the fluorinated amino acid side chain seen at -14 hydroxymethyl, C-15 hydroxy or C-20 demethylation is attached to the maytansinoid compound, and wherein The mercaptan or disulfide bond functional group of the mercapto group of the deuterated amino acid side chain is located at a carbon atom having one or two substituents, which are CH 3 , C 2 H 5 , and have 1 to 10 a linear alkyl or alkenyl group of a carbon atom, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocycloalkyl group And furthermore, one of the substituents may be H, and wherein the fluorenyl group has a linear chain length of at least three carbon atoms between the carbonyl functional group and the sulfur atom.

本發明之較佳結合物為包含與式(VIII)之美登素類化合物結合之抗-CD38抗體38SB13、38SB18、38SB19、38SB30、38SB31或38SB39或其同源物或片段之結合物： 其中：Y1'表示(CR7R8)l(CR9=CR10)p(C≡C)qAr(CR5R6)mDu(CR11=CR12)r(C≡C)sBt(CR3R4)nCR1R2S-，其中：A、B及D各自獨立地為具有3-10個碳原子之環烷基或環烯基、簡單或經取代之芳基或雜環芳族基團或雜環烷基；R3、R4、R5、R6、R7、R8、R9、R11及R12各自獨立地為H、CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基；且l、m、n、o、p、q、r、s及t各自獨立地為0或1至5之整數，其限制條件為l、m、n、o、p、q、r、s及t中之至少兩者在任何時候不均為零。 A preferred combination of the invention is a combination comprising an anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 or a homolog or fragment thereof in combination with a maytansinoid compound of formula (VIII): Where: Y 1 ' represents (CR 7 R 8 ) l (CR 9 =CR 10 ) p (C≡C) q A r (CR 5 R 6 ) m D u (CR 11 =CR 12 ) r (C≡C s B t (CR 3 R 4 ) n CR 1 R 2 S-, wherein: A, B and D are each independently a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, simple or substituted An aryl or heterocyclic aromatic group or a heterocycloalkyl group; R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 11 and R 12 are each independently H, CH 3 , C 2 H 5 , a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a hetero group a cycloaromatic group or a heterocycloalkyl group; and l, m, n, o, p, q, r, s, and t are each independently 0 or an integer from 1 to 5, with the constraints of l, m, n At least two of o, p, q, r, s, and t are not zero at all times.

較佳地，R1為H且R2為甲基或R1及R2為甲基。 Preferably, R 1 is H and R 2 is methyl or R 1 and R 2 are methyl.

本發明之甚至更佳之結合物為包含與式(IX-L)、(IX-D)或(IX-D,L)之美登素類化合物結合之抗-CD38抗體38SB13、38SB18、 38SB19、38SB30、38SB31或38SB39或其同源物或片段之結合物： 其中：Y1表示(CR7R8)l(CR5R6)m(CR3R4)nCR1R2S-，其中：R1及R2各自獨立地為CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基、雜環芳族基團或雜環烷基，且此外R2可為H；R3、R4、R5、R6、R7及R8各自獨立地為H、CH3、C2H5、具有1至10個碳原子之直鏈烷基或烯基、具有3至10個碳原子之支鏈或環狀烷基或烯基、苯基、經取代之苯基或雜環芳族基團或雜環烷基；l、m及n各自獨立地為1至5之整數，且此外n可為0；且May表示在C-3、C-14羥甲基、C-15羥基或C-20去甲基處帶有側鏈之美登醇。 An even more preferred combination of the invention is an anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30 comprising a combination of a genus of the formula (IX-L), (IX-D) or (IX-D, L). Combination of 38SB31 or 38SB39 or a homolog or fragment thereof: Wherein: Y 1 represents (CR 7 R 8 ) l (CR 5 R 6 ) m (CR 3 R 4 ) n CR 1 R 2 S-, wherein: R 1 and R 2 are each independently CH 3 , C 2 H 5 , a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group, a heterocyclic aromatic group a group or a heterocycloalkyl group, and further R 2 may be H; R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently H, CH 3 , C 2 H 5 , having 1 to a linear alkyl or alkenyl group of 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aromatic group or a heterocyclic ring Alkyl; l, m and n are each independently an integer from 1 to 5, and further n may be 0; and May represents C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20. The melamine with a side chain at the base.

式(IX-L)、(IX-D)及(IX-D,L)之較佳實施例包括以下式(IX-L)、(IX-D)及(IX-D,L)之化合物，其中：R1為H且R2為甲基或R1及R2為甲基，R1為H，R2為甲基，R5、R6、R7及R8各自為H；l及m各自為1；n為0，R1及R2為甲基；R5、R6、R7及R8各自為H；l及m為1；n為0。 Preferred embodiments of the formulae (IX-L), (IX-D) and (IX-D, L) include the compounds of the following formulae (IX-L), (IX-D) and (IX-D, L), Wherein: R 1 is H and R 2 is methyl or R 1 and R 2 are methyl, R 1 is H, R 2 is methyl, and R 5 , R 6 , R 7 and R 8 are each H; m is each 1; n is 0, R 1 and R 2 are methyl; R 5 , R 6 , R 7 and R 8 are each H; l and m are 1; n is 0.

較佳地，細胞毒性劑由式(IX-L)表示。 Preferably, the cytotoxic agent is represented by the formula (IX-L).

本發明之另一較佳結合物為包含與式(X)之美登素類化合物結合 之抗-CD38抗體38SB13、38SB18、38SB19、38SB30、38SB31或38SB39或其同源物或片段之結合物： 其中取代基係如上文針對式(IX)所定義。 Another preferred combination of the invention is a combination comprising an anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 or a homolog or fragment thereof in combination with a maytansinoid compound of formula (X): Wherein the substituent is as defined above for formula (IX).

尤其較佳者為其中R1為H，R2為甲基，R5、R6、R7及R8各自為H，l及m各自為1，且n為0之上述化合物中之任一者。 Particularly preferred are any of the above compounds wherein R 1 is H, R 2 is methyl, R 5 , R 6 , R 7 and R 8 are each H, and l and m are each 1, and n is 0. By.

其他尤其較佳者為其中R1及R2為甲基，R5、R6、R7、R8各自為H，l及m為1，且n為0之上述化合物中之任一者。 Other particularly preferred are any of the above compounds wherein R 1 and R 2 are methyl, R 5 , R 6 , R 7 and R 8 are each H, 1 and m are 1, and n is 0.

此外，L-胺基醯基立體異構體為較佳。 Further, an L-amino thiol stereoisomer is preferred.

2004年5月20日申請之申請中之美國專利申請案第10/849,136號中所教示之美登素類化合物中之各者亦可用於本發明之細胞毒性結合物。美國專利申請案第10/849,136號之全部揭示內容以引用的方式併入本文中。 Each of the maytansinoids taught in U.S. Patent Application Serial No. 10/849,136, filed on May 20, 2004, is also incorporated herein by reference. The entire disclosure of U.S. Patent Application Serial No. 10/849,136 is incorporated herein by reference.

含雙硫鍵之連接基團Linker group containing a disulfide bond

為使美登素類化合物與諸如38SB13、38SB18、38SB19、38SB30、38SB31或38SB39抗體之細胞結合劑連接，美登素類化合物包含連接部分。連接部分含有允許完全活性美登素類化合物在特定位點處釋放之化學鍵。合適之化學鍵在此項技術中係熟知的且包括雙硫 鍵、酸不穩定鍵、光不穩定鍵、肽酶不穩定鍵及酯酶不穩定鍵。較佳為雙硫鍵。 In order to link the maytansinoid compound to a cell binding agent such as 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibody, the maytansinoid compound comprises a linking moiety. The linking moiety contains a chemical bond that allows the complete active maytansinoid compound to be released at a particular site. Suitable chemical linkages are well known in the art and include disulfide A bond, an acid labile bond, a photolabile bond, a peptidase labile bond, and an esterase labile bond. Preferred is a disulfide bond.

連接部分亦包含反應性化學基團。在一較佳實施例中，反應性化學基團可經由雙硫鍵連接部分與美登素類化合物共價結合。 The linking moiety also contains reactive chemical groups. In a preferred embodiment, the reactive chemical group can be covalently bound to the maytansinoid via a disulfide linkage moiety.

尤其較佳之反應性化學基團為N-琥珀醯亞胺酯及N-磺基琥珀醯亞胺酯。 Particularly preferred reactive chemical groups are N-succinimide and N-sulfosuccinimide.

包含含有反應性化學基團之連接部分之尤其較佳之美登素類化合物為美登醇及其類似物之C-3酯，其中連接部分含有雙硫鍵且化學反應性基團包含N-琥珀醯亞胺酯或N-磺基琥珀醯亞胺酯。 Particularly preferred maytansinoids comprising a linking moiety containing a reactive chemical group are C-3 esters of maytansinol and analogs thereof, wherein the linking moiety contains a disulfide bond and the chemically reactive group comprises N-amber Yttrium imidate or N-sulfosuccinimide.

美登素類化合物上之許多位置可充當與連接部分化學連接之位置。舉例而言，預期具有羥基之C-3位置、經羥甲基修飾之C-14位置、經羥基修飾之C-15位置及具有羥基之C-20位置均適用。然而，C-3位置為較佳且美登醇之C-3位置尤其較佳。 Many positions on the maytansinoid compound serve as a site for chemical attachment to the linking moiety. For example, it is expected that a C-3 position having a hydroxyl group, a C-14 position modified with a hydroxymethyl group, a C-15 position modified with a hydroxyl group, and a C-20 position having a hydroxyl group are applicable. However, the C-3 position is preferred and the C-3 position of maytansinol is particularly preferred.

儘管具有連接部分之美登醇之酯的合成係根據含雙硫鍵之連接部分來描述，但熟習此項技術者應瞭解具有其他化學鍵(如上文所述)之連接部分亦可用於本發明，同樣可使用其他美登素類化合物。其他化學鍵之特定實例包括酸不穩定鍵、光不穩定鍵、肽酶不穩定鍵及酯酶不穩定鍵。併入本文中之美國專利第5,208,020號之揭示內容教示帶有該等鍵之美登素類化合物的產生。 Although the synthesis of the maytansyl ester having a linking moiety is described in terms of a linking moiety containing a disulfide bond, those skilled in the art will appreciate that linking moieties having other chemical linkages (as described above) can also be used in the present invention, as well. Other maytansinoid compounds can be used. Specific examples of other chemical bonds include acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds. The disclosure of U.S. Pat.

具有帶有反應性基團之雙硫鍵部分之美登素類化合物及美登素類化合物衍生物的合成描述於美國專利第6,441,163號及第6,333,410號及美國申請案第10/161,651號中，該等文獻之各者均以引用的方式併入本文中。 The synthesis of a maytansinoid compound having a disulfide bond moiety having a reactive group and a derivative of the maytansinoid compound is described in U.S. Patent Nos. 6,441,163 and 6,333,410, and U.S. Patent Application Serial No. 10/161,651. Each of the documents is incorporated herein by reference.

含反應性基團之美登素類化合物(諸如DM1)與諸如38SB13、38SB18、38SB19、38SB30、38SB31或38SB39抗體之抗體反應而產生細胞毒性結合物。此等結合物可藉由HPLC或由凝膠過濾來純化。 A reactive group-containing maytansinoid such as DM1 reacts with an antibody such as 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibody to produce a cytotoxic conjugate. These conjugates can be purified by HPLC or by gel filtration.

用於產生此等抗體-美登素類化合物結合物之數種極佳流程提供於美國專利第6,333,410號及美國申請案第09/867,598號、第10/161,651號及第10/024,290號中，該等文獻之各者之全文均併入本文中。 Several excellent procedures for the production of such antibody-maytansinoid conjugates are provided in U.S. Patent No. 6,333,410 and U.S. Application Serial Nos. 09/867,598, 10/161,651 and 10/024,290. The entire text of each of these documents is incorporated herein.

一般而言，可將抗體於水性緩衝液中之溶液與莫耳過量之具有帶有反應性基團之雙硫鍵部分的美登素類化合物一起培育。可藉由添加過量胺(諸如乙醇胺、牛磺酸等)來中止反應混合物。接著可藉由凝膠過濾來純化美登素類化合物-抗體結合物。 In general, a solution of the antibody in an aqueous buffer can be incubated with a molar excess of a maytansinoid compound having a disulfide bond moiety having a reactive group. The reaction mixture can be stopped by the addition of an excess of an amine such as ethanolamine, taurine or the like. The maytansinoid-antibody conjugate can then be purified by gel filtration.

每個抗體分子所結合之美登素類化合物分子數可藉由以分光光度法量測252nm與280nm下之吸光度比值來測定。平均每個抗體分子結合1-10個美登素類化合物分子為較佳。 The number of molecules of the maytansin compound to which each antibody molecule is bound can be determined by spectrophotometric measurement of the absorbance ratio at 252 nm to 280 nm. It is preferred that each antibody molecule binds to 1-10 maytansinoid molecules per molecule.

可對抗體與美登素類化合物藥物之結合物活體外抑制各種不合需要之細胞株增殖之能力進行評估。舉例而言，諸如人類淋巴瘤細胞株Daudi、人類淋巴瘤細胞株Ramos、人類多發性骨髓瘤細胞株MOLP-8及人類急性T淋巴細胞性白血病株系MOLT-4之細胞株可易用於評估此等化合物之細胞毒性。可將待評估之細胞暴露於化合物歷時24小時且藉由已知方法在直接檢定中量測細胞之存活分數。接著可自檢定結果計算IC50值。 The ability of the combination of the antibody and the maytansinoid drug to inhibit the proliferation of various undesirable cell lines in vitro can be evaluated. For example, cell lines such as human lymphoma cell line Daudi, human lymphoma cell line Ramos, human multiple myeloma cell line MOLP-8, and human acute T lymphocytic leukemia strain MOLT-4 can be easily used for evaluation. Cytotoxicity of these compounds. The cells to be evaluated can be exposed to the compound for 24 hours and the survival fraction of the cells can be measured in a direct assay by known methods. The IC 50 value can then be calculated from the assay results.

含PEG之連接基團PEG-containing linking group

如美國申請案第10/024,290號中所陳述，美登素類化合物亦可使用PEG連接基團與細胞結合劑連接。此等PEG連接基團可溶於水中與非水性溶劑中，且可用於連接一或多種細胞毒性劑與細胞結合劑。例示性PEG連接基團包括雜雙官能PEG連接子，其在連接子之相對端經由官能性氫硫基，或在一端經由雙硫基而在另一端經由活性酯與細胞毒性劑及細胞結合劑結合。 The maytansinoid compounds can also be linked to a cell binding agent using a PEG linking group as set forth in U.S. Patent Application Serial No. 10/024,290. These PEG linking groups are soluble in water and non-aqueous solvents and can be used to link one or more cytotoxic agents to cell binding agents. Exemplary PEG linking groups include heterobifunctional PEG linkers via a functional thiol group at the opposite end of the linker, or via a disulfide group at one end and an cytotoxic agent and cell binder at the other end. Combine.

作為使用PEG連接基團合成細胞毒性結合物之一般實例，再次參 考美國申請案第10/024,290號之特定細節。合成以一或多種帶有反應性PEG部分之細胞毒性劑與細胞結合劑之反應起始，使得各反應性PEG部分之末端活性酯由細胞結合劑之胺基酸殘基置換，以得到包含一或多種與細胞結合劑經PEG連接基團共價鍵結之細胞毒性劑之細胞毒性結合物。 As a general example of the synthesis of cytotoxic conjugates using PEG linking groups, see again Specific details of US Application No. 10/024,290. Synthesis begins with the reaction of one or more cytotoxic agents with a reactive PEG moiety with a cell binding agent such that the terminal active ester of each reactive PEG moiety is replaced by an amino acid residue of the cell binding agent to provide a Or a plurality of cytotoxic conjugates of a cytotoxic agent covalently bonded to the cell binding agent via a PEG linking group.

紫杉烷Taxane

本發明之細胞毒性結合物中所用之細胞毒性劑亦可為紫杉烷或其衍生物。 The cytotoxic agent used in the cytotoxic conjugate of the present invention may also be a taxane or a derivative thereof.

紫杉烷為包括太平洋紫杉醇(紫杉醇)(一種細胞毒性天然產物)及多烯紫杉醇(紫杉德(Taxotere))(一種半合成衍生物)之化合物家族，此兩種化合物在癌症治療中廣泛使用。紫杉烷為抑制微管蛋白解聚而致使細胞死亡之有絲分裂紡錘體毒劑。儘管多烯紫杉醇及太平洋紫杉醇為治療癌症之適用藥劑，但其抗腫瘤活性因其對正常細胞之非特異性毒性而受到限制。此外，如太平洋紫杉醇及多烯紫杉醇本身之化合物並不充分有效以細胞結合劑之結合物之形式使用。 Taxanes are a family of compounds including paclitaxel (paclitaxel), a cytotoxic natural product, and docetaxel (a taxonomic derivative), which are widely used in cancer therapy. . A taxane is a mitotic spindle poison that inhibits the depolymerization of tubulin and causes cell death. Although docetaxel and paclitaxel are suitable agents for the treatment of cancer, their antitumor activity is limited by their non-specific toxicity to normal cells. Furthermore, compounds such as paclitaxel and docetaxel itself are not sufficiently effective to be used in the form of a combination of cell binding agents.

用於製備細胞毒性結合物之較佳紫杉烷為式(XI)之紫杉烷： A preferred taxane for the preparation of a cytotoxic conjugate is a taxane of formula (XI):

用於合成可在本發明之細胞毒性結合物中使用之紫杉烷之方法連同用於使紫杉烷與諸如抗體之細胞結合劑結合之方法一起詳細描述於美國專利第5,416,064號、第5,475,092號、第6,340,701號、第 6,372,738號及第6,436,931號及美國申請案第10/024,290號、第10/144,042號、第10/207,814號、第10/210,112號及第10/369,563號中。 A method for synthesizing a taxane which can be used in the cytotoxicity conjugate of the present invention, together with a method for binding a taxane to a cell binding agent such as an antibody, is described in detail in U.S. Patent Nos. 5,416,064 and 5,475,092. , No. 6,340,701, No. 6,372,738 and 6,436,931, and U.S. Application Serial Nos. 10/024,290, 10/144,042, 10/207,814, 10/210,112, and 10/369,563.

茅屋黴素衍生物Tortoise derivative

本發明之細胞毒素亦可為茅屋黴素衍生物。茅屋黴素衍生物為吡咯并[1,4]苯并二氮呯(PBD)，其為藉由共價結合DNA小凹槽中之鳥嘌呤之N2來發揮其生物特性之一類已知化合物。PBD包括許多小凹槽結合劑，諸如安麯黴素(anthramycin)、新茴黴素(neothramycin)及DC-81。 The cytotoxin of the present invention may also be a tomaymycin derivative. The tomaymycin derivative is pyrrolo[1,4]benzodiazepine (PBD), which is a known compound that exerts its biological properties by covalently binding N2 of guanine in the small groove of DNA. PBD includes many small groove binders such as anthramycin, neothramycin, and DC-81.

保留高細胞毒性且可與細胞結合劑有效連接之新穎茅屋黴素衍生物描述於國際申請案第PCT/IB2007/000142號中，該申請案之內容以引用的方式併入本文中。細胞結合劑-茅屋黴素衍生物複合物允許茅屋黴素衍生物之全測度細胞毒性作用以靶向方式僅施加於不合需要之細胞，由此避免因損害非靶向健康細胞所引起之副作用。 A novel tomaymycin derivative that retains high cytotoxicity and is operably linked to a cell binding agent is described in International Application No. PCT/IB2007/000142, the disclosure of which is incorporated herein by reference. The cell-binding agent-maumycin derivative complex allows the full-measure cytotoxic effect of the tomaymycin derivative to be applied only to undesirable cells in a targeted manner, thereby avoiding side effects caused by damage to non-targeted healthy cells.

本發明之細胞毒性劑包含一或多種經由連接基團與諸如38SB13、38SB18、38SB19、38SB30、38SB31或38SB39抗體之細胞結合劑連接之茅屋黴素衍生物。連接基團為經由習知方法與茅屋黴素衍生物共價結合之化學部分之一部分。在一較佳實施例中，該化學部分可經由雙硫鍵與茅屋黴素衍生物共價結合。 The cytotoxic agent of the present invention comprises one or more tomaymycin derivatives linked via a linking group to a cell binding agent such as 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibodies. The linking group is part of a chemical moiety that is covalently bound to the tomaymycin derivative by conventional methods. In a preferred embodiment, the chemical moiety can be covalently bound to the tomaymycin derivative via a disulfide bond.

適用於本發明之茅屋黴素衍生物具有下文所展示之式(XII)： 其中： ----表示一可選單鍵；表示一單鍵或一雙鍵；其限制條件為當─表示一單鍵時，相同或不同之U及U'獨立地表示H，且相同或不同之W及W'係獨立地選自由以下各基團組成之群：OH、諸如-OR之醚、諸如-OCOR之酯(例如乙酸酯)、諸如-OCOOR之碳酸酯、諸如-OCONRR'之胺基甲酸酯、環狀胺基甲酸酯(使得N10及C11為環之一部分)、諸如-NRCONRR'之脲、諸如-OCSNHR之硫代胺基甲酸酯、環狀硫代胺基甲酸酯(使得N10及C11為環之一部分)、-S H、諸如-S R之硫醚、諸如-S O R之亞碸、諸如-SOOR之碸、諸如-SO3-之磺酸酯、諸如-NRSOOR之磺醯胺、諸如-NRR'之胺、可選擇之環胺(使得N10及C11為環之一部分)、諸如-NROR'之胲衍生物、諸如-NRCOR之醯胺、諸如-N3之疊氮基、氰基、鹵基、三烷基鏻或三芳基鏻、衍生自胺基酸之基團；較佳地，W及W'相同或不同且為OH、Ome、Oet、NHCONH2、SMe；且當─表示一雙鍵時，U及U'不存在且W及W'表示H；R1、R2、R1'、R2'相同或不同且係獨立地選自鹵離子或視情況經一或多個Hal、CN、NRR'、CF3、OR、芳基、Het、S(O)qR取代之烷基，或R1與R2及R1'與R2'分別一起形成含雙鍵基團=B及=B'。 The tomaymycin derivative suitable for use in the present invention has the formula (XII) shown below: Where: ---- indicates an optional single button; Representing a single bond or a double bond; the restriction is that when a single bond is indicated, the same or different U and U' independently represent H, and the same or different W and W' are independently selected from the following Groups of groups: OH, ethers such as -OR, esters such as -OCOR (eg acetate), carbonates such as -OCOOR, urethanes such as -OCONRR', cyclic carbamic acid An ester (so that N10 and C11 are part of a ring), a urea such as -NRCONRR', a thiocarbamate such as -OCSNHR, a cyclic thiocarbamate (so that N10 and C11 are part of a ring) , -SH, a thioether such as -SR, a hydrazine such as -SOR, a hydrazine such as -SOOR, a sulfonate such as -SO 3 -, a sulfonamide such as -NRSOOR, an amine such as -NRR', the selected cyclic amine (such that N10 and C11 is part of a ring), such as -NROR 'of the hydroxylamine derivatives of Amides such as -NRCOR, an azido such as -N 3, the cyano group, a halogen group, a trialkyl phosphonium Or a triaryl fluorene, a group derived from an amino acid; preferably, W and W' are the same or different and are OH, Ome, Oet, NHCONH 2 , SMe; and when ─ represents a double bond, U and U 'Does not exist and W and W' indicate H R1, R2, R1 ', R2 ' are the same or different and are independently selected halides or optionally substituted with one or more Hal, CN, NRR ', CF 3, OR, aryl, Het, S (O) q R substituted alkyl, or R1 and R2 and R1' and R2' together form a double bond-containing group = B and = B', respectively.

較佳地，R1與R2及R1'與R2'分別一起形成含雙鍵基團=B及=B'。 Preferably, R1 and R2 and R1' and R2' together form a double bond-containing group = B and = B', respectively.

B及B'相同或不同且係獨立地選自視情況經一或多個Hal、CN、NRR'、CF3、OR、芳基、Het、S(O)qR取代之烯基，或B及B'表示氧原子。 B and B 'are the same or different and are independently selected optionally substituted with one or more Hal, CN, NRR', CF 3, OR, aryl group, the substituted Het, S (O) q R alkenyl group, or B And B' represents an oxygen atom.

較佳地，B=B'。 Preferably, B = B'.

更佳地，B=B'==CH2或=CH-CH3。 More preferably, B = B' = = CH 2 or = CH - CH 3 .

X、X'相同或不同且係獨立地選自一或多個-O-、-NR-、-(C=O)-、-S(O)q-。 X, X' are the same or different and are independently selected from one or more of -O-, -NR-, -(C=O)-, -S(O) q- .

較佳地，X=X'。 Preferably, X = X'.

更佳地，X=X'=O。 More preferably, X = X' = O.

A、A'相同或不同且係獨立地選自視情況含有氧、氮或硫原子之烷基或烯基，其各自視情況經一或多個Hal、CN、NRR'、CF3、OR、S(O)qR、芳基、Het、烷基、烯基取代。 A, A 'are the same or different and are independently selected alkyl optionally containing an oxygen, nitrogen or sulfur atoms or an alkenyl group, each optionally substituted with one or more Hal, CN, NRR', CF 3, OR, S(O) q R, aryl, Het, alkyl, alkenyl substituted.

較佳地，A=A'。 Preferably, A = A'.

更佳地，A=A'=直鏈未經取代之烷基。 More preferably, A = A' = a linear unsubstituted alkyl group.

Y、Y'相同或不同且係獨立地選自H、OR；較佳地，Y=Y'。 Y, Y' are the same or different and are independently selected from H, OR; preferably, Y = Y'.

更佳地，Y=Y'=烷氧基，更佳為甲氧基。 More preferably, Y = Y' = alkoxy, more preferably methoxy.

T為-NR-，-O-，-S(O)q-，或各自視情況經一或多個Hal、CN、NRR'、CF3、R、OR、S(O)qR及/或連接子取代之4至10員芳基、環烷基、雜環或雜芳基，或視情況經一或多個Hal、CN、NRR'、CF3、OR、S(O)qR及/或連接子取代之支鏈烷基，或經一或多個Hal、CN、NRR'、CF3、OR、S(O)qR及/或連接子取代之直鏈烷基。 T is -NR-, -O-, -S(O) q -, or each of them optionally passes one or more of Hal, CN, NRR', CF 3 , R, OR, S(O) q R and/or linker of 4-10 substituted aryl, cycloalkyl, heterocyclyl or heteroaryl, or optionally substituted with one or more Hal, CN, NRR ', CF 3, oR, S (O) q R and / the linker or unsubstituted branched alkyl, or with one or more Hal, CN, NRR ', CF 3, oR, S (O) q R and / or linker of the substituted linear alkyl group.

較佳地，T為視情況經一或多個連接子取代之4至10員芳基或雜芳基，更佳為苯基或吡啶基。 Preferably, T is a 4 to 10 membered aryl or heteroaryl group optionally substituted with one or more linkers, more preferably a phenyl or pyridyl group.

該連接子包含連接基團。合適之連接基團在此項技術中係熟知的且包括硫醇、硫醚、雙硫基、硫醚基、酸不穩定基團、光不穩定基團、肽酶不穩定基團及酯酶不穩定基團。較佳為雙硫基及硫醚基。 The linker comprises a linking group. Suitable linking groups are well known in the art and include thiols, thioethers, disulfo groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterases. Unstable group. Preferred are dithio groups and thioether groups.

當連接基團為含硫醇-、硫醚(-S-)或雙硫基(-S-S-)之基團時，帶有硫醇、硫醚或雙硫基之側鏈可為直鏈或支鏈，芳族或雜環的。一般技術者可易於鑑別合適之側鏈。 When the linking group is a group containing a thiol-, thioether (-S-) or a dithio group (-SS-), the side chain having a thiol, a thioether or a disulfide group may be a straight chain or Branched, aromatic or heterocyclic. One of ordinary skill can readily identify suitable side chains.

較佳地，該連接子具有下式：-G-D-(Z)p-S-Z'其中： G為一單鍵或雙鍵、-O-、-S-或-NR-；D為一單鍵或-E-、-E-NR-、-E-NR-F-、-E-O-、-E-O-F-、-E-NR-CO-、-E-NR-CO-F-、-E-CO-、-CO-E-、-E-CO-F、-E-S-、-E-S-F-、-E-NR-C-S-、-E-NR-CS-F-；其中E及F相同或不同且係獨立地選自直鏈或支鏈-(OCH2CH2)i烷基(OCH2CH2)j-、-烷基(OCH2CH2)i-烷基-、-(OCH2CH2)i-、-(OCH2CH2)i環烷基(OCH2CH2)j-、-(OCH2CH2)i雜環(OCH2CH2)j-、-(OCH2CH2)i芳基(OCH2CH2)j-、-(OCH2CH2)i雜芳基(OCH2CH2)j-、-烷基-(OCH2CH2)i烷基(OCH2CH2)j-、-烷基-(OCH2CH2)i-、-烷基-(OCH2CH2)i環烷基(OCH2CH2)j-、-烷基(OCH2CH2)i雜環(OCH2CH2)j-、-烷基-(OCH2CH2)i芳基(OCH2CH2)j-、-烷基(OCH2CH2)i雜芳基(OCH2CH2)j-、-環烷基-烷基-、-烷基-環烷基-、-雜環-烷基-、-烷基-雜環-、-烷基-芳基-、-芳基-烷基-、-烷基-雜芳基-、-雜芳基-烷基-；其中相同或不同之i及j為整數且係獨立地選自0、1至2000；Z為直鏈或支鏈-烷基-；p為0或1；Z'表示H、諸如COR之硫醇保護基、R20或SR20，其中R20表示H、甲基、烷基、視情況經取代之環烷基、芳基、雜芳基或雜環，其限制條件為當Z'為H時，該化合物與由藉由添加硫醇基-SH至PBD部分之一之亞胺鍵-NH=上而引起之分子內環化所形成之相應化合物平衡。 Preferably, the linker has the formula: -GD-(Z) p -S-Z' wherein: G is a single bond or a double bond, -O-, -S- or -NR-; D is a single Key or -E-, -E-NR-, -E-NR-F-, -EO-, -EOF-, -E-NR-CO-, -E-NR-CO-F-, -E-CO -, -CO-E-, -E-CO-F, -ES-, -ESF-, -E-NR-CS-, -E-NR-CS-F-; where E and F are the same or different and are Independently selected from linear or branched -(OCH 2 CH 2 ) ialkyl (OCH 2 CH 2 ) j -, -alkyl (OCH 2 CH 2 ) i -alkyl-, -(OCH 2 CH 2 ) i -, -(OCH 2 CH 2 ) i cycloalkyl (OCH 2 CH 2 ) j -, -(OCH 2 CH 2 ) i heterocyclic ring (OCH 2 CH 2 ) j -, -(OCH 2 CH 2 ) i Aryl (OCH 2 CH 2 ) j -, -(OCH 2 CH 2 ) i heteroaryl (OCH 2 CH 2 ) j -, -alkyl-(OCH 2 CH 2 ) i alkyl (OCH 2 CH 2 ) j -, -alkyl-(OCH 2 CH 2 ) i -, -alkyl-(OCH 2 CH 2 ) i cycloalkyl (OCH 2 CH 2 ) j -, -alkyl (OCH 2 CH 2 ) i Ring (OCH 2 CH 2 ) j -, -alkyl-(OCH 2 CH 2 ) i aryl (OCH 2 CH 2 ) j -, -alkyl (OCH 2 CH 2 ) i heteroaryl (OCH 2 CH 2 ) j -, -cycloalkyl-alkyl-, -alkyl-cycloalkyl-, -heterocyclo-alkyl-, -alkyl-heterocyclic-, -alkyl-aryl-, -aryl- Alkyl-, -alkyl-hetero -, -heteroaryl-alkyl-; wherein i or j which are the same or different are integers and are independently selected from 0, 1 to 2000; Z is a linear or branched-alkyl group; p is 0 or 1 ;Z' represents H, a thiol protecting group such as COR, R20 or SR20, wherein R20 represents H, methyl, alkyl, optionally substituted cycloalkyl, aryl, heteroaryl or heterocyclic, which is limited The condition is that when Z' is H, the compound is in equilibrium with the corresponding compound formed by intramolecular cyclization caused by the addition of the thiol group-SH to the imine bond -NH= of one of the PBD moieties.

相同或不同之n、n'為0或1。 The same or different n, n' is 0 or 1.

q為0、1或2。 q is 0, 1, or 2.

R、R'相同或不同且係獨立地選自H、烷基、芳基，其各自視情況經Hal、CN、NRR'、CF3、R、OR、S(O)qR、芳基、Het取代； 或其醫藥學上可接受之鹽、水合物或水合鹽，或此等化合物或其光學異構體、外消旋體、非對映體或對映異構體之多晶型結晶結構。 R, R 'are the same or different and are independently selected from H, alkyl, aryl, each optionally substituted with Hal, CN, NRR', CF 3, R, OR, S (O) q R, aryl, Het substituted; or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystal of such a compound or an optical isomer, racemate, diastereomer or enantiomer thereof structure.

具有幾何異構體及立體異構體之通式(XII)之化合物亦為本發明之一部分。 Compounds of the formula (XII) having geometric isomers and stereoisomers are also part of the invention.

已知式(XII)之茅屋黴素衍生物之N-10、C-11雙鍵在水、醇、硫醇、一級胺或二級胺、脲及其他親核試劑存在下可易於以可逆方式轉化成相應亞胺加合物。此過程為可逆的且可易於在脫水劑存在下，在非質子性有機溶劑中，在真空中或在高溫下再生相應茅屋黴素衍生物(Z.Tozuka,1983,J.Antibiotics,36：276)。 It is known that the N-10 and C-11 double bonds of the tomaymycin derivative of the formula (XII) can be easily reversibly in the presence of water, alcohol, thiol, primary or secondary amine, urea and other nucleophiles. Conversion to the corresponding imine adduct. This process is reversible and can easily regenerate the corresponding tomaymycin derivative in an aprotic organic solvent in a vacuum or at elevated temperature in the presence of a dehydrating agent (Z. Tozuka, 1983, J. Antibiotics , 36 :276 ).

因此，通式(XIII)之茅屋黴素衍生物之可逆衍生物亦可用於本發明： 其中A、X、Y、n、T、A'、X'、Y'、n'、R1、R2、R1'、R2'係如式(XII)中所定義，且W、W'相同或不同且係選自由以下各基團組成之群：OH、諸如-OR之醚、諸如-OCOR之酯(例如乙酸酯)、-COOR、諸如-OCOOR之碳酸酯、諸如-OCONRR'之胺基甲酸酯、環狀胺基甲酸酯(使得N10及C11為環之一部分)、諸如-NRCONRR'之脲、諸如-OCSNHR之硫代胺基甲酸酯、環狀硫代胺基甲酸酯(使得N10及C11為環之一部分)、-SH、諸如-SR之硫醚、諸如-SOR之亞碸、諸如-SOOR之碸、諸如-SO3-之磺酸酯、諸如-NRSOOR之磺醯胺、諸如-NRR'之胺、可選擇之環胺(使得N10及C11為環之一部分)、諸如-NROR'之胲衍生物、諸如-NRCOR之醯胺、-NRCONRR'、諸如-N3之疊氮基、氰 基、鹵基、三烷基鏻或三芳基鏻、衍生自胺基酸之基團。較佳地，W及W'相同或不同且為OH、Ome、Oet、NHCONH2、SMe。 Therefore, a reversible derivative of the tomaymycin derivative of the formula (XIII) can also be used in the present invention: Wherein A, X, Y, n, T, A', X', Y', n', R1, R2, R1', R2' are as defined in formula (XII), and W, W' are the same or different And is selected from the group consisting of OH, an ether such as -OR, an ester such as -OCOR (such as acetate), -COOR, a carbonate such as -OCOOR, an amine group such as -OCONRR' An acid ester, a cyclic urethane (so that N10 and C11 are part of a ring), a urea such as -NRCONRR', a thiocarbamate such as -OCSNHR, a cyclic thiocarbamate ( Let N10 and C11 be a part of the ring), -SH, a thioether such as -SR, a hydrazine such as -SOR, a hydrazine such as -SOOR, a sulfonate such as -SO 3 -, a sulfonamide such as -NRSOOR An amine such as -NRR', an optional cyclic amine (so that N10 and C11 are part of a ring), an anthracene derivative such as -NROR', a guanamine such as -NRCOR, -NRCONRR', a stack such as -N 3 Nitro, cyano, halo, trialkyl or triaryl fluorene, a group derived from an amino acid. Preferably, W and W' are the same or different and are OH, Ome, Oet, NHCONH 2 , SMe.

式(XIII)化合物因此可被視為溶劑合物，當溶劑為水時，其可被視為包括水之溶劑合物；此等溶劑合物可尤其適用。 The compound of the formula (XIII) can therefore be regarded as a solvate, and when the solvent is water, it can be regarded as a solvate comprising water; such solvates can be especially suitable.

在一較佳實施例中，本發明之茅屋黴素衍生物係選自由以下各物組成之群：8,8'-[1,3-苯二基雙(亞甲氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] In a preferred embodiment, the tomaymycin derivative of the present invention is selected from the group consisting of: 8,8'-[1,3-phenyldiylbis(methyleneoxy)]-double [ (S)-2-Ethylene-(E)-yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo Diazin-5-one]

8,8'-[5-甲氧基-1,3-苯二基雙(亞甲氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[5-Methoxy-1,3-phenyldiylbis(methyleneoxy)]-bis[(S)-2-iethylene-(E)-yl-7-methoxy -1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[1,5-戊烷二基雙(氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[1,5-pentanediylbis(oxy)]-bis[(S)-2-i-(E)-yl-7-methoxy-1,2,3, 11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[1,4-丁烷二基雙(氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[1,4-butanediylbis(oxy)]-bis[(S)-2-i-(E)-yl-7-methoxy-1,2,3, 11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[3-甲基-1,5-戊烷二基雙(氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[3-Methyl-1,5-pentanediylbis(oxy)]-bis[(S)-2-iethylene-(E)-yl-7-methoxy-1 , 2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[2,6-吡啶二基雙(氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[2,6-Pyridinylbis(oxy)]-bis[(S)-2-iB-(E)-yl-7-methoxy-1,2,3,11a -tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[4-(3-第三丁氧基羰基胺基丙氧基)-2,6-吡啶二基雙(亞甲氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[4-(3-Tertioxycarbonylaminopropoxy)-2,6-pyridinediylbis(methyleneoxy)]-bis[(S)-2-A -(E)-yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[5-(3-胺基丙氧基)-1,3-苯二基雙(亞甲氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[5-(3-Aminopropyloxy)-1,3-phenyldiylbis(methyleneoxy)]-bis[(S)-2-i-(E)-yl -7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[5-(N-甲基-3-第三丁氧基羰基胺基丙基)-1,3-苯二基雙(亞甲氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1- c][1,4]苯并二氮呯-5-酮] 8,8'-[5-(N-methyl-3-tributoxycarbonylaminopropyl)-1,3-phenyldiylbis(methyleneoxy)]-bis[(S)- 2-ethylidene-(E)-yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1- c][1,4]benzodiazepine-5-one]

8,8'-{5-[3-(4-甲基-4-甲基二硫基-戊醯基胺基)丙氧基]-1,3-苯二基雙(亞甲氧基)}-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-{5-[3-(4-Methyl-4-methyldithio-pentanylamino)propoxy]-1,3-phenyldiylbis(methyleneoxy) }-bis[(S)-2-Ethylene-(E)-yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1, 4] benzodiazepine-5-one]

8,8'-[5-乙醯基硫基甲基-1,3-苯二基雙(亞甲氧基)]-雙[(S)-2-亞甲基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[5-Ethylthiomethyl-1,3-phenyldiylbis(methyleneoxy)]-bis[(S)-2-methylene-7-methoxy- 1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

雙-{2-[(S)-2-亞甲基-7-甲氧基-5-側氧基-1,3,,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-8-基氧基]-乙基}-胺基甲酸第三丁酯 Bis-{2-[(S)-2-methylene-7-methoxy-5-oxooxy-1,3,,11a-tetrahydro-5H-pyrrolo[2,1-c][ 1,4]benzodiazepine-8-yloxy]-ethyl}-carbamic acid tert-butyl ester

8,8'-[3-(2-乙醯基硫基乙基)-1,5-戊烷二基雙(氧基)]-雙[(S)-2-亞甲基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[3-(2-Ethylthioethyl)-1,5-pentanediylbis(oxy)]-bis[(S)-2-methylene-7-- Oxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[5-(N-4-巰基-4,4-二甲基丁醯基)胺基-1,3-苯二基雙(亞甲氧基)]-雙[7-甲氧基-2-亞甲基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[5-(N-4-Mercapto-4,4-dimethylbutanyl)amino-1,3-phenyldiylbis(methyleneoxy)]-bis[7-methoxy -2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[5-(N-4-甲基二硫基-4,4-二甲基丁醯基)-胺基-1,3-苯二基雙(亞甲氧基)]-雙[7-甲氧基-2-亞甲基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[5-(N-4-methyldithio-4,4-dimethylbutanyl)-amino-1,3-phenyldiylbis(methyleneoxy)]-double [ 7-Methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[5-(N-甲基-N-(2-巰基-2,2-二甲基乙基)胺基-1,3-苯二基(亞甲氧基)]-雙[7-甲氧基-2-亞甲基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[5-(N-Methyl-N-(2-mercapto-2,2-dimethylethyl)amino-1,3-phenyldiyl(methyleneoxy)]-double [7-Methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[5-(N-甲基-N-(2-甲基二硫基-2,2-二甲基乙基)胺基-1,3-苯二基(亞甲氧基)]-雙[7-甲氧基-2-亞甲基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[5-(N-Methyl-N-(2-methyldithio-2,2-dimethylethyl)amino-1,3-phenyldiyl (methyleneoxy) )]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine- 5-ketone]

8,8'-[(4-(2-(4-巰基-4-甲基)-戊醯胺基-乙氧基-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(4-(2-(4-Mercapto-4-methyl)-pentylamino-ethoxy-pyridine-2,6-dimethyl)-dioxy]-double [ (S)-2-Ethylene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzoic Nitroin-5-one]

8,8'-[(1-(2-(4-甲基-4-甲基二硫基)-戊醯胺基-乙氧基)-苯-3,5-二甲 基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(1-(2-(4-methyl-4-methyldithio)-pentylamino-ethoxy)-benzene-3,5-dimethyl ))-dioxy]-bis[(S)-2-iB-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1- c][1,4]benzodiazepine-5-one]

8,8'-[(4-(3-(4-甲基-4-甲基二硫基)-戊醯胺基-丙氧基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(4-(3-(4-methyl-4-methyldithio)-pentylamino-propoxy)-pyridine-2,6-dimethyl)-dioxo ]]-bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1, 4] benzodiazepine-5-one]

8,8'-[(4-(4-(4-甲基-4-甲基二硫基)-戊醯胺基-丁氧基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(4-(4-(4-Methyl-4-methyldithio)-pentylamino-butoxy)-pyridine-2,6-dimethyl)-dioxo ]]-bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1, 4] benzodiazepine-5-one]

8,8'-[(4-(3-[4-(4-甲基-4-甲基二硫基-戊醯基)-哌嗪-1-基]-丙基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(4-(3-[4-(4-Methyl-4-methyldithio-pentanyl)-piperazin-1-yl]-propyl)-pyridine-2, 6-Dimethyl)-dioxy]-bis[(S)-2-i-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[ 2,1-c][1,4]benzodiazepine-5-one]

8,8'-[(1-(3-[4-(4-甲基-4-甲基二硫基-戊醯基)-哌嗪-1-基]-丙基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(1-(3-[4-(4-Methyl-4-methyldithio-pentanyl)-piperazin-1-yl]-propyl)-benzene-3, 5-dimethyl)-dioxy]-bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[ 2,1-c][1,4]benzodiazepine-5-one]

8,8'-[(4-(2-{2-[2-(4-甲基-4-甲基二硫基-戊醯基胺基)-乙氧基]-乙氧基}-乙氧基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(4-(2-{2-[2-(4-Methyl-4-methyldithio-pentylamino)-ethoxy]-ethoxy}-B Oxy)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)-2-i-(E)-yl-7-dimethoxy-1,2,3, 11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[(1-(2-{2-[2-(2-{2-[2-(4-甲基-4-甲基二硫基-戊醯基胺基)-乙氧基]-乙氧基}-乙氧基)-乙氧基]-乙氧基}-乙氧基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(1-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldithio-pentanylamino)-ethoxy ]]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)- 2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine-5 -ketone]

8,8'-[(1-(2-{2-[2-(4-甲基-4-甲基二硫基-戊醯基胺基)-乙氧基]-乙氧基}-乙氧基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(1-(2-{2-[2-(4-Methyl-4-methyldithio-pentylamino)-ethoxy]-ethoxy}-B Oxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-i-(E)-yl-7-dimethoxy-1,2,3, 11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

8,8'-[(4-(2-{2-[2-(2-{2-[2-(4-甲基-4-甲基二硫基-戊醯基胺基)-乙氧基]-乙氧基}-乙氧基)-乙氧基]-乙氧基}-乙氧基)-吡啶-2,6-二甲基)- 二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(4-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldithio-pentanylamino)-ethoxy ]]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-pyridine-2,6-dimethyl)- Dioxy]-bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][ 1,4] benzodiazepine-5-one]

8,8'-[(1-(2-[甲基-(2-甲基-2-甲基二硫基-丙基)-胺基]-乙氧基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(1-(2-[Methyl-(2-methyl-2-methyldithio-propyl)-amino]-ethoxy)-benzene-3,5-di Methyl)-dioxy]-bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1 -c][1,4]benzodiazepine-5-one]

8,8'-[(4-(3-[甲基-(4-甲基-4-甲基二硫基-戊醯基)-胺基]-丙基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(4-(3-[Methyl-(4-methyl-4-methyldithio-pentanyl)-amino]-propyl)-pyridine-2,6-di Methyl)-dioxy]-bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1 -c][1,4]benzodiazepine-5-one]

8,8'-[(4-(3-[甲基-(2-甲基-2-甲基二硫基-丙基)-胺基]-丙基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(4-(3-[Methyl-(2-methyl-2-methyldithio-propyl)-amino]-propyl)-pyridine-2,6-dimethyl ))-dioxy]-bis[(S)-2-iB-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1- c][1,4]benzodiazepine-5-one]

8,8'-[(1-(4-甲基-4-甲基二硫基)-戊醯胺基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(1-(4-Methyl-4-methyldithio)-pentylamino)-benzene-3,5-dimethyl)-dioxy]-bis[(S -2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine -5-ketone]

以及相應巰基衍生物，或其醫藥學上可接受之鹽、水合物或水合鹽，或此等化合物或其光學異構體、外消旋體、非對映體或對映異構體之多晶型結晶結構。 And corresponding thiol derivatives, or pharmaceutically acceptable salts, hydrates or hydrated salts thereof, or such compounds or optical isomers, racemates, diastereomers or enantiomers thereof Crystalline crystal structure.

較佳化合物為以下式之化合物： 或 其中X、X'、A、A'、Y、Y'、T、n、n'係如上文所定義。 Preferred compounds are those of the formula: or Wherein X, X', A, A', Y, Y', T, n, n' are as defined above.

式(XII)化合物可以熟習此項技術者熟知之許多方式來製備。該等化合物可(例如)藉由應用或調整下文所述之方法，或如熟習此項技術者所瞭解之其變體來合成。適當之修改及替代對於熟習此項技術者將顯而易見且為其所熟知或可易於自科學文獻獲得。詳言之，該等方法可見於R.C.Larock,Comprehensive Organic Transformations,Wiley-VCH Publishers,1999中。 Compounds of formula (XII) can be prepared in a number of ways well known to those skilled in the art. Such compounds can be synthesized, for example, by applying or modifying the methods described below, or variants as would be understood by those skilled in the art. Appropriate modifications and substitutions will be apparent to and will be readily apparent to those skilled in the art. In particular, such methods can be found in RC Larock, Comprehensive Organic Transformations , Wiley-VCH Publishers, 1999.

用於合成可在本發明中使用之茅屋黴素衍生物之方法描述於國際申請案第PCT/IB2007/000142號中。本發明之化合物可由多種合成途徑來製備。試劑及起始物質可購得，或易於由一般技術者以熟知技術來合成(例如參見WO 00/12508、WO 00/12507、WO 2005/040170、WO 2005/085260、FR1516743，M.Mori等人，1986,Tetrahedron,42：3793-3806)。 A method for synthesizing a tomaymycin derivative which can be used in the present invention is described in International Application No. PCT/IB2007/000142. The compounds of the invention can be prepared by a variety of synthetic routes. Reagents and starting materials are commercially available or can be readily synthesized by the skilled artisan using well known techniques (see, for example, WO 00/12508, WO 00/12507, WO 2005/040170, WO 2005/085260, FR 1516743, M. Mori et al. , 1986, Tetrahedron , 42 : 3793-3806).

本發明之結合物分子可使用任何技術來形成。本發明之茅屋黴素衍生物可經由酸不穩定連接子或經由光不穩定連接子與抗體或其他細胞結合劑連接。該等衍生物可與具有合適序列之肽縮合且隨後與細胞結合劑連接以產生肽酶不穩定連接子。可製備含有一級羥基之結合物，其可經琥珀醯化且與細胞結合劑連接以產生可由細胞內酯酶裂解以釋放游離衍生物之結合物。較佳地，所合成之衍生物含有游離或經保護之硫醇基，且接著一或多種含雙硫鍵或硫醇之衍生物各自經由雙硫鍵或硫醚鍵與細胞結合劑共價連接。 The conjugate molecules of the invention can be formed using any technique. The tomaymycin derivative of the invention may be linked to an antibody or other cell binding agent via an acid labile linker or via a photolabile linker. The derivatives can be condensed with a peptide having the appropriate sequence and subsequently ligated with a cell binding agent to produce a peptidase labile linker. A conjugate comprising a primary hydroxyl group can be prepared which can be amber sulphated and linked to a cell binding agent to produce a conjugate that can be cleaved by intracellular esterase to release the free derivative. Preferably, the synthesized derivative contains a free or protected thiol group, and then one or more disulfide- or thiol-containing derivatives are each covalently linked to the cell binding agent via a disulfide bond or a thioether bond. .

眾多結合方法教示於USP 5,416,064及USP 5,475,092中。茅屋黴素衍生物可經修飾以產生游離胺基且接著經由酸不穩定連接子或光不穩定連接子與抗體或其他細胞結合劑連接。具有游離胺基或羧基之茅屋黴素衍生物可與肽縮合且隨後與細胞結合劑連接以產生肽酶不穩定連接子。在連接子上具有游離羥基之茅屋黴素衍生物可經琥珀醯化且 與細胞結合劑連接以產生可由細胞內酯酶裂解以釋放游離藥物之結合物。最佳地，茅屋黴素衍生物經處理以產生游離或經保護之硫醇基，且接著含雙硫鍵或硫醇之茅屋黴素二聚體經由雙硫鍵與細胞結合劑連接。 A number of combinations are taught in USP 5,416,064 and USP 5,475,092. The tomaymycin derivative can be modified to produce a free amine group and then attached to the antibody or other cell binding agent via an acid labile linker or a photolabile linker. A tomaymycin derivative having a free amine or carboxyl group can be condensed with a peptide and subsequently linked to a cell binding agent to produce a peptidase labile linker. A tomaymycin derivative having a free hydroxyl group on the linker can be amber-stained and Attached to the cell binding agent to produce a conjugate that can be cleaved by intracellular esterase to release the free drug. Most preferably, the tomaymycin derivative is treated to produce a free or protected thiol group, and then the tomays dimer containing a disulfide bond or a thiol is attached to the cell binding agent via a disulfide bond.

較佳地，單株抗體或細胞結合劑-茅屋黴素衍生物結合物為如上所述經由雙硫鍵連接且能夠傳遞茅屋黴素衍生物之結合物。該等細胞結合結合物可藉由已知方法來製備，諸如藉由用琥珀醯亞胺基吡啶基-二硫代丙酸酯(SPDP)修飾單株抗體來製備(Carlsson等人，1978,Biochem.J.,173：723-737)。接著將所得硫代吡啶基藉由用含硫醇之茅屋黴素衍生物處理來置換以產生雙硫鍵連接之結合物。或者，在芳基二硫基-茅屋黴素衍生物之狀況下，細胞結合結合物之形成可藉由以預先引入抗體分子中之氫硫基直接置換茅屋黴素衍生物之芳基-硫醇來實現。易於藉由任一種方法來製備含有1至10個經由雙硫橋連接之茅屋黴素衍生藥物之結合物。 Preferably, the monoclonal antibody or cell binding agent-maumycin derivative conjugate is a conjugate that is linked via a disulfide bond as described above and capable of transmitting a tomaymycin derivative. Such cell-binding conjugates can be prepared by known methods, such as by modification of monoclonal antibodies with amber iminopyridyl-dithiopropionate (SPDP) (Carlsson et al., 1978, Biochem). .J ., 173 :723-737). The resulting thiopyridyl group is then replaced by treatment with a thiol-containing tomaymycin derivative to produce a disulfide-linked conjugate. Alternatively, in the case of an aryldithio-maurimycin derivative, the formation of a cell-binding conjugate can be achieved by directly replacing the aryl-thiol of the tomaymycin derivative with a thiol group previously introduced into the antibody molecule. to realise. It is easy to prepare a conjugate comprising 1 to 10 tomaymycin-derived drugs linked via a disulfide bridge by any method.

更特定言之，2.5mg/ml濃度之經二硫基-硝基吡啶基修飾之抗體於含有2mM EDTA之0.05M磷酸鉀緩衝液(pH 7.5)中之溶液用含硫醇之茅屋黴素衍生物(1.3莫耳當量/二硫吡啶基)處理。以分光光度法在325nm監測硫基-硝基吡啶自經修飾抗體之釋放，該釋放在約16小時內完成。藉由凝膠過濾經Sephadex G-25或Sephacryl S300管柱純化抗體-茅屋黴素衍生物結合物且使其不含未反應之藥物及其他低分子量物質。每個抗體分子所結合之茅屋黴素衍生物部分之數目可藉由測量在230nm與275nm之吸光比值來測定。藉由此方法每個抗體分子可經由雙硫鍵連接平均1-10個茅屋黴素衍生物分子。 More specifically, a solution of a dithio-nitropyridyl modified antibody at a concentration of 2.5 mg/ml in a 0.05 M potassium phosphate buffer (pH 7.5) containing 2 mM EDTA was derived from a thiol-containing tomaymycin. Treatment (1.3 mol/dithiopyridyl). The release of the thio-nitropyridine from the modified antibody was monitored spectrophotometrically at 325 nm and the release was completed in about 16 hours. The antibody-maumycin derivative conjugate was purified by gel filtration through a Sephadex G-25 or Sephacryl S300 column and was free of unreacted drugs and other low molecular weight substances. The number of portions of the tomaymycin derivative to which each antibody molecule is bound can be determined by measuring the absorbance ratio at 230 nm to 275 nm. By this method, each antibody molecule can be linked to an average of 1-10 tomaymycin derivative molecules via a disulfide bond.

結合對於針對抗原表現細胞之結合親和力的影響可使用先前Liu等人，1996,Proc.Natl.Acad.Sci.U.S.A.,93：8618-8623所述之方法來測定。茅屋黴素衍生物及其抗體結合物對於細胞株之細胞毒性可藉由 Goldmacher等人，1985,J.Immunol.,135：3648-3651所述之細胞增殖曲線之反向外推來測量。此等化合物對黏附細胞株之細胞毒性可藉由Goldmacher等人，1986,J.Cell Biol.,102：1312-1319所述之細胞群落檢定來測定。 The effect of binding on binding affinity to antigen-expressing cells can be determined using the method described previously by Liu et al., 1996, Proc . Natl . Acad . Sci . USA ., 93 : 8618-8623. The cytotoxicity of the tomaymycin derivative and its antibody conjugate to the cell line can be measured by the inverse extrapolation of the cell proliferation curve described by Goldmacher et al., 1985, J. Immunol ., 135 : 3648-3651. The cytotoxicity of such compounds to adherent cell lines can be determined by cell population assays as described by Goldmacher et al., 1986, J. Cell Biol ., 102 : 1312-1319.

細黴素衍生物Leptomycin derivative

本發明之細胞毒素亦可為細黴素衍生物。根據本發明，"細黴素衍生物"係指Kalesse等人(2002,Synthesis 8：981-1003)中所定義之細黴素家族之成員，包括：細黴素，諸如細黴素A及細黴素B；卡爾他汀(callystatin)；瑞佳酮(ratjadone)，諸如瑞佳酮A及瑞佳酮B；安桂黴素(anguinomycin)，諸如安桂黴素A、B、C、D；卡蘇黴素(kasusamycin)；萊托他汀(leptolstatin)；萊託福甯(leptofuranin)，諸如萊託福寧A、B、C、D。細黴素A及B之衍生物為較佳。 The cytotoxin of the present invention may also be a leptomycin derivative. According to the invention, "leomycin derivative" means a member of the family of leptomycins as defined in Kalesse et al. (2002, Synthesis 8 : 981-1003), including: leptomycin, such as leptomycin A and fine Streptomycin B; calstatin; ratjadone, such as ruthenone A and ruthenone B; anguinomycin, such as aguimycin A, B, C, D; (kasusamycin); leptolstatin; leptofuranin, such as letofotin A, B, C, D. Derivatives of leptomycin A and B are preferred.

更特定言之，本發明之衍生物具有式(I)： 其中：Ra及Ra'為H或-Alk；較佳地，Ra為-Alk，尤其為甲基，且Ra'為H；R17為視情況經OR、CN、NRR'、全氟烷基取代之烷基；較佳地，R17為烷基，更佳為甲基或乙基；R9為視情況經OR、CN、NRR'、全氟烷基取代之烷基；較佳地，R9為烷基，更佳為甲基；X為-O-或-NR-；較佳地，X為-NR-； Y為-U-、-NR-U-、-O-U-、-NR-CO-U-、-U-NR-CO-、-U-CO-、-CO-U-；較佳地，當X為-O-時，Y為-U-、-NR-U-、-U-NR-CO-；其中U係選自直鏈或支鏈-Alk-、-Alk(OCH2CH2)m-、-(OCH2CH2)m-Alk-、-Alk-(OCH2CH2)m-Alk-、-(OCH2CH2)m-、-環烷基-、-雜環-、-環烷基-Alk-、-Alk-環烷基-、-雜環-Alk-、-Alk-雜環-；其中m為選自1至2000之整數；較佳地，U為直鏈或支鏈-Alk-；Z為-Alk-；n為0或1；較佳地，n為0；T表示H、諸如Ac之硫醇保護基、R1或SR1，其中R1表示H、甲基、Alk、環烷基、視情況取代之芳基或雜環；或T表示： 其中：Ra、Ra'、R17、R9、X、Y、Z、n係如上文所定義；較佳地，T為H或SR1，其中R1表示Alk，更佳為甲基；相同或不同之R、R'為H或烷基；Alk表示直鏈或支鏈烷基；較佳地，Alk表示(-(CH2-q(CH3)q)p-，其中p表示1至10之整數；且q表示0至2之整數；較佳地，Alk表示-(CH2)-或-C(CH3)2-；或其醫藥學上可接受之鹽、水合物或水合鹽，或此等化合物或其光學異構體、外消旋體、非對映體或對映異構體之多晶型結晶結 構。 More specifically, the derivative of the present invention has the formula (I): Wherein: Ra and Ra' are H or -Alk; preferably, Ra is -Alk, especially methyl, and Ra' is H; R17 is optionally substituted by OR, CN, NRR', perfluoroalkyl An alkyl group; preferably, R17 is an alkyl group, more preferably a methyl group or an ethyl group; and R9 is an alkyl group optionally substituted by OR, CN, NRR', perfluoroalkyl group; preferably, R9 is an alkyl group. More preferably, it is a methyl group; X is -O- or -NR-; preferably, X is -NR-; Y is -U-, -NR-U-, -OU-, -NR-CO-U- , -U-NR-CO-, -U-CO-, -CO-U-; preferably, when X is -O-, Y is -U-, -NR-U-, -U-NR- CO-; wherein U is selected from linear or branched -Alk -, - Alk (OCH 2 CH 2) m -, - (OCH 2 CH 2) m -Alk -, - Alk- (OCH 2 CH 2) m -Alk-, -(OCH 2 CH 2 ) m -, -cycloalkyl-, -heterocyclic-, -cycloalkyl-Alk-, -Alk-cycloalkyl-, -heterocyclic-Alk-, -Alk -heterocycle-; wherein m is an integer selected from 1 to 2000; preferably, U is a straight or branched chain -Alk-; Z is -Alk-; n is 0 or 1; preferably, n is 0 ; T represents H, a thiol protecting group such as Ac, R 1 or SR 1 , wherein R 1 represents H, methyl, Alk, cycloalkyl, optionally substituted aryl or heterocyclic; or T represents: Wherein: Ra, Ra', R17, R9, X, Y, Z, n are as defined above; preferably, T is H or SR 1 , wherein R 1 represents Alk, more preferably methyl; the same or different R, R' is H or alkyl; Alk represents a linear or branched alkyl group; preferably, Alk represents (-(CH 2-q (CH 3 ) q ) p -, wherein p represents 1 to 10 An integer; and q represents an integer from 0 to 2; preferably, Alk represents -(CH 2 )- or -C(CH 3 ) 2 -; or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or Polymorphic crystalline structures of such compounds or their optical isomers, racemates, diastereomers or enantiomers.

較佳化合物可選自以下各物：(2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-甲基硫基-乙基)-醯胺 Preferred compounds may be selected from the group consisting of (2E, 10E, 12E, 16Z, 18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptyl-19- ((2S,3S)-3-methyl-6-oxooxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy-19-carbon-2,10,12 ,16,18-pentenoic acid (2-methylthio-ethyl)-decylamine

雙-[(2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-巰基乙基)-醯胺] Bis-[(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptyl-19-((2S,3S)- 3-methyl-6-tertiaryoxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy-nine-carbon-2,10,12,16,18-pentene Acid (2-mercaptoethyl)-decylamine]

(2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-巰基-乙基)-醯胺 (2E, 10E, 12E, 16Z, 18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptyl-19-((2S,3S)-3-A Keto-6-o-oxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy-nine-carbon-2,10,12,16,18-pentenoic acid 2-mercapto-ethyl)-guanamine

(2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-甲基二硫基-乙基)-醯胺 (2E, 10E, 12E, 16Z, 18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptyl-19-((2S,3S)-3-A Keto-6-o-oxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy-nine-carbon-2,10,12,16,18-pentenoic acid 2-methyldithio-ethyl)-guanamine

(2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-甲基-2-甲基二硫基-丙基)-醯胺 (2E, 10E, 12E, 16Z, 18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptyl-19-((2S,3S)-3-A Keto-6-o-oxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy-nine-carbon-2,10,12,16,18-pentenoic acid 2-methyl-2-methyldithio-propyl)-guanamine

(2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-巰基-2-甲基-丙基)-醯胺 (2E, 10E, 12E, 16Z, 18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptyl-19-((2S,3S)-3-A Keto-6-o-oxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy-nine-carbon-2,10,12,16,18-pentenoic acid 2-mercapto-2-methyl-propyl)-guanamine

或其醫藥學上可接受之鹽、水合物或水合鹽，或此等化合物或其光學異構體、外消旋體、非對映體或對映異構體之多晶型結晶結構。 Or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystalline structure of such a compound or an optical isomer, racemate, diastereomer or enantiomer thereof.

為使衍生物與細胞結合劑連接，衍生物須包括允許衍生物經由諸如雙硫鍵、硫醚鍵、酸不穩定基團、光不穩定基團、肽酶不穩定基團或酯酶不穩定基團之鍵聯與細胞結合劑連接之部分(連接基團)。衍 生物經製備成使其含有使細黴素衍生物經由(例如)雙硫鍵、硫醚鍵、酸不穩定基團、光不穩定基團、肽酶不穩定基團或酯酶不穩定基團與細胞結合劑連接所必需之部分。為另外增強於水溶液中之溶解性，連接基團可含有聚乙二醇間隔基。較佳地，使用硫醚或雙硫鍵，因為靶分子之還原環境可使硫醚或雙硫鍵裂解且在細胞毒性相關聯增加之情況下釋放衍生物。 In order for a derivative to be attached to a cell binding agent, the derivative must include allowing the derivative to be unstable via, for example, a disulfide bond, a thioether bond, an acid labile group, a photolabile group, a peptidase labile group, or an esterase. A moiety (linking group) to which a bond of a group is attached to a cell binding agent. Yan The organism is prepared to contain a leptomycin derivative via, for example, a disulfide bond, a thioether bond, an acid labile group, a photolabile group, a peptidase labile group, or an esterase labile group. The part necessary for attachment to a cell binding agent. To additionally enhance solubility in aqueous solutions, the linking group may contain polyethylene glycol spacers. Preferably, a thioether or disulfide bond is used because the reducing environment of the target molecule can cleave the thioether or disulfide bond and release the derivative with increased cytotoxicity.

本發明之化合物可藉由多種合成途徑來製備。試劑及起始物質可購得，或易於由一般技術者以熟知技術來合成。用於合成可在本發明之細胞毒性結合物中使用之細黴素衍生物之方法連同用於使該等細黴素衍生物與諸如抗體之細胞結合劑結合之方法一起詳細描述於歐洲專利申請案第06290948.6號中，該申請案之內容以引用的方式併入本文中。 The compounds of the invention can be prepared by a variety of synthetic routes. Reagents and starting materials are commercially available or can be readily synthesized by the skilled artisan using well known techniques. A method for synthesizing a leptomycin derivative which can be used in the cytotoxicity conjugate of the present invention, together with a method for binding such a leptomycin derivative to a cell binding agent such as an antibody, is described in detail in the European Patent Application The contents of this application are incorporated herein by reference.

CC-1065類似物CC-1065 analogue

本發明之細胞毒性結合物中所用之細胞毒性劑亦可為CC-1065或其衍生物。 The cytotoxic agent used in the cytotoxic conjugate of the present invention may also be CC-1065 or a derivative thereof.

CC-1065為自澤倫氏鏈黴菌(Streptomyces zelensis)之培養肉湯分離之有效抗腫瘤抗生素。CC-1065之活體外效用比諸如阿黴素(doxorubicin)、甲胺喋呤(methotrexate)及長春新鹼(vincristine)之常用抗癌藥物大約1000倍(B.K.Bhuyan等人，1982,Cancer Res.,42,3532-3537)。CC-1065及其類似物揭示於美國專利第6,372,738號、第6,340,701號、第5,846,545號及第5,585,499號中。 CC-1065 is an effective anti-tumor antibiotic isolated from the culture broth of Streptomyces zelensis . The in vitro efficacy of CC-1065 is approximately 1000 times higher than that of commonly used anticancer drugs such as doxorubicin, methotrexate, and vincristine (BKBhuyan et al., 1982, Cancer Res ., 42 , 3532-3537). </ RTI><RTIgt;</RTI><RTIgt;

CC-1065之細胞毒性效能與其烷基化活性及其DNA結合或DNA插入活性相關聯。此等兩種活性存在於分子之獨立部分中。因此，烷基化活性含於環丙基吡咯并吲哚(cyclopropapyrroloindole，CPI)子單位中且DNA結合活性存在於兩個吡咯并吲哚子單位中。 The cytotoxic potency of CC-1065 is related to its alkylation activity and its DNA binding or DNA insertion activity. These two activities are present in separate parts of the molecule. Thus, the alkylation activity is contained in the cyclopropapyrroloindole (CPI) subunit and the DNA binding activity is present in the two pyrroindoxidin units.

儘管CC-1065作為細胞毒性劑具有某些具有吸引力之特徵，但其 在治療用途中存有侷限性。將CC-1065投予小鼠引起緩發性肝中毒而在單次靜脈內給予12.5μg/kg後第50天產生死亡率(V.L.Reynolds等人，1986,J.Antibiotics,XXIX：319-334)。此已激勵努力研發不引起緩發毒性之類似物，且已描述以CC-1065為模型之較簡單類似物之合成(M.A.Warpehoski等人，1988,J.Med.Chem.,31：590-603)。 Although CC-1065 has some attractive characteristics as a cytotoxic agent, it has limitations in therapeutic use. Administration of CC-1065 to mice caused delayed onset liver toxicity and mortality occurred on day 50 after a single intravenous administration of 12.5 μg/kg (VL Reynolds et al., 1986, J. Antibiotics , XXIX : 319-334). This has motivated efforts to develop analogs that do not cause delayed toxicity, and has described the synthesis of simpler analogs modeled on CC-1065 (MA Warrphoski et al., 1988, J. Med . Chem ., 31 : 590-603). .

在另一系列之類似物中，CPI部分經環丙基苯并吲哚(cyclopropabenzindole，CBI)部分置換(D.L.Boger等人，1990,J.Org.Chem.,55：5823-5833；D.L.Boger等人，1991,BioOrg.Med.Chem.Lett.,1：115-120)。此等化合物維持母體藥物之高活體外效能，而在小鼠體內不引起緩發毒性。如同CC-1065，此等化合物為以共價方式與DNA之小凹槽結合而引起細胞死亡之烷基化劑。然而，最具前景之類似物阿多來新(Adozelesin)及卡折來新(Carzelesin)之臨床評估得到令人失望之結果(B.F.Foster等人，1996,Investigational New Drugs,13：321-326；I.Wolff等人，1996,Clin.Cancer Res.,2：1717-1723)。此等藥物因其高全身毒性而顯示不良治療作用。 In another series of analogs, the CPI moiety is partially replaced by cyclopropabenzindole (CBI) (DL Boger et al, 1990, J. Org. Chem. , 55 : 5823-5833; DL Boger et al, 1991, BioOrg . Med . Chem . Lett ., 1 : 115-120). These compounds maintain the high in vitro potency of the parent drug without causing delayed toxicity in the mouse. Like CC-1065, these compounds are alkylating agents that cause cell death by binding to small grooves of DNA in a covalent manner. However, the clinical evaluation of the most promising analogues, Adozelesin and Carzelesin, has disappointing results (BFFoster et al., 1996, Investigational New Drugs , 13 :321-326; .Wolff et al., 1996, Clin . Cancer Res ., 2 :1717-1723). These drugs show adverse therapeutic effects due to their high systemic toxicity.

CC-1065類似物之治療功效可藉由經靶向傳遞至腫瘤位點改變活體內分布，從而對非靶向組織產生較低毒性，且由此降低全身毒性而極大地改良。為達成此目的，已描述CC-1065之類似物及衍生物與特異性靶向腫瘤細胞之細胞結合劑之結合物(美國專利第5,475,092號、第5,585,499號、第5,846,545號)。此等結合物通常在活體外顯示高標靶特異性細胞毒性，且在小鼠中之人類腫瘤異種移植模型中顯示優越之抗腫瘤活性(R.V.J.Chari等人，1995,Cancer Res.,55：4079-4084)。 The therapeutic efficacy of the CC-1065 analog can be greatly improved by targeted delivery to the tumor site to alter in vivo distribution, resulting in less toxicity to non-targeted tissues, and thereby reducing systemic toxicity. To this end, combinations of analogs and derivatives of CC-1065 with cell binding agents that specifically target tumor cells have been described (U.S. Patent Nos. 5,475,092, 5,585,499, 5,846,545). These conjugates typically exhibit high target-specific cytotoxicity in vitro and exhibit superior anti-tumor activity in human tumor xenograft models in mice (RV J Chari et al, 1995, Cancer Res ., 55 :4079- 4084).

最近，已描述在水介質中具有增強溶解性之CC-1065類似物之前藥(歐洲專利申請案第06290379.4號)。在此等前藥中，分子之烷基化部分之酚基經官能基保護，該官能基使藥物在儲存於酸性水溶液之過程中穩定，且賦予藥物與未經保護之類似物相比增加之水溶性。保護 基易於在活體內在生理pH值下裂解而得到相應活性藥物。在EP 06290379.4中所述之前藥中，酚取代基經保護成在生理pH值下具有電荷之含磺酸之胺基甲酸苯酯，且由此具有增強之水溶性。為另外增強水溶性，可將可選擇之聚乙二醇間隔基引入吲哚基子單位與諸如雙硫基之可裂解鍵之間的連接子中。此間隔基的引入不改變藥物之效能。 Recently, a CC-1065 analog prodrug having enhanced solubility in an aqueous medium has been described (European Patent Application No. 06290379.4). In such prodrugs, the phenolic group of the alkylated portion of the molecule is protected by a functional group that stabilizes the drug during storage in the acidic aqueous solution and imparts an increase in the drug compared to the unprotected analog. Water soluble. protection The base is easily cleaved in vivo at physiological pH to give the corresponding active drug. In the prodrugs described in EP 06290379.4, the phenolic substituent is protected as a sulfonic acid-containing phenyl carbamate having a charge at physiological pH and thus has enhanced water solubility. To additionally enhance water solubility, an optional polyethylene glycol spacer can be introduced into the linker between the indenyl unit and the cleavable bond such as a disulfide group. The introduction of this spacer does not alter the efficacy of the drug.

用於合成可在本發明之細胞毒性結合物中使用之CC-1065類似物之方法連同用於使該等類似物與諸如抗體之細胞結合劑結合之方法一起詳細描述於EP 06290379.4(其內容以引用的方式併入本文中)及美國專利第5,475,092號、第5,846,545號、第5,585,499號、第6,534,660號及第6,586,618號及美國申請案第10/116,053號及第10/265,452號中。 Methods for synthesizing CC-1065 analogs that can be used in the cytotoxic conjugates of the invention are described in detail in EP 06290379.4 together with methods for combining such analogs with cell binding agents such as antibodies (the contents of which are Citations are incorporated herein by reference to U.S. Patent Nos. 5,475,092, 5,846,545, 5,585,499, 6,534,660 and 6,586,618, and U.S. Application Serial Nos. 10/116,053 and 10/265,452.

其他藥物Other drugs

諸如甲胺喋呤、道諾黴素(daunorubicin)、阿黴素、長春新鹼、長春鹼(vinblastine)、美法侖(melphalan)、絲裂黴素C(mitomycin C)、苯丁酸氮芥(chlorambucil)、卡奇黴素(calicheamicin)、托布來新(tubulysin)及托布來新類似物、多卡黴素(duocarmycin)及多卡黴素類似物、海兔毒素及海兔毒素類似物之藥物亦適用於製備本發明之結合物。藥物分子亦可經諸如血清白蛋白之中間載體分子與抗體分子連接。如(例如)美國申請案第09/740991號中所述之多克黴素(Doxarubicin)及單諾黴素(Darorubicin)化合物亦可為適用之細胞毒性劑。 Such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil Similar to (chlorambucil), calicheamicin, tubulysin and toboidin analogs, duocarmycin and docamycin analogues, dolastatin and dolastatin The medicaments are also suitable for the preparation of the combinations of the invention. The drug molecule can also be linked to the antibody molecule via an intermediate carrier molecule such as serum albumin. Doxarubicin and Darorubicin compounds as described in, for example, U.S. Application Serial No. 09/740,991, may also be suitable cytotoxic agents.

治療組合物Therapeutic composition

本發明亦係關於一種用於治療哺乳動物之過度增生性病症或發炎疾病或自體免疫疾病之治療組合物，其包含治療有效量之本發明之化合物及醫藥學上可接受之載劑。在一實施例中，該醫藥組合物係用於治療癌症，其包括(但不限於)以下各病症：癌瘤，包括膀胱、乳房、結腸、腎、肝、肺、卵巢、胰腺、胃、子宮頸、甲狀腺及皮膚之 癌瘤；包括鱗狀細胞癌；淋巴系之造血腫瘤，包括白血病、急性淋巴細胞性白血病、急性淋巴母細胞白血病、B細胞淋巴瘤、T細胞淋巴瘤、伯基特氏淋巴瘤；骨髓系之造血腫瘤，包括急性及慢性骨髓性白血病及前髓細胞性白血病；間葉源性腫瘤，包括纖維肉瘤及橫紋肌肉瘤；其他腫瘤，包括黑素瘤、精原細胞瘤、畸胎癌、神經母細胞瘤及神經膠質瘤；中樞及周邊神經系統之腫瘤，包括星形細胞瘤、神經母細胞瘤、神經膠質瘤及神經鞘瘤；間葉源性腫瘤，包括纖維肉瘤、橫紋肌肉瘤及骨肉瘤；及其他腫瘤，包括黑素瘤、著色性乾皮病、角化棘皮瘤、精原細胞瘤、甲狀腺濾泡癌及畸胎癌；及仍待確定之其中CD38優勢表現之其他癌症。在一較佳實施例中，本發明之醫藥組合物係用於治療其中表現CD38之癌症，諸如非霍奇金氏淋巴瘤、霍奇金氏淋巴瘤、毛細胞白血病、多發性骨髓瘤、慢性淋巴細胞性白血病、慢性骨髓性白血病、急性骨髓性白血病或急性淋巴細胞性白血病，及仍待確定之其中CD38優勢表現之其他癌症。在另一實施例中，本發明之醫藥組合物可用於治療自體免疫疾病，諸如全身性紅斑性狼瘡症、類風濕性關節炎、多發性硬化症、克隆氏病、潰瘍性結腸炎、胃炎、橋本氏甲狀腺炎、強直性脊椎炎、C型肝炎相關之冷凝球蛋白性血管炎、慢性局竈性腦炎、大疱性類天疱瘡、A型血友病、膜性增生性絲球體腎炎、修格蘭氏症候群、成人及青少年皮肌炎、成人多發性肌炎、慢性蕁麻疹、原發性膽汁性肝硬化症、特發性血小板減少性紫癜、視神經脊髓炎、格雷氏甲狀腺機能障礙疾病、大疱性類天疱瘡、膜性增生性絲球體腎炎、徹奇-斯全司症候群及哮喘。在另一實施例中，該醫藥組合物係關於其他病症，諸如移植排斥反應，諸如腎移植排斥反應、肝移植排斥反應、肺移植排斥反應、心臟移植排斥反應及骨髓移植排斥反應；移植物抗宿主疾病；病毒感染，諸如mV感染、HIV感染、AIDS等；及寄生蟲感染，諸如梨形鞭毛蟲症、變形 蟲症、血吸蟲病；及如由一般技術者所確定之其他病症。 The invention also relates to a therapeutic composition for treating a hyperproliferative disorder or an inflammatory or autoimmune disorder in a mammal comprising a therapeutically effective amount of a compound of the invention and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition is for treating cancer, including but not limited to the following conditions: cancer, including bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, son Cervical, thyroid, and skin Carcinoma; including squamous cell carcinoma; lymphoid hematopoietic tumors, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; Hematopoietic tumors, including acute and chronic myelogenous leukemia and promyelocytic leukemia; mesenchymal tumors, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblasts Tumors and gliomas; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; Other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma; and other cancers still to be identified with CD38 superiority. In a preferred embodiment, the pharmaceutical composition of the present invention is for treating a cancer in which CD38 is expressed, such as non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, multiple myeloma, chronic Lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia or acute lymphocytic leukemia, and other cancers still to be identified in which CD38 is superior. In another embodiment, the pharmaceutical composition of the present invention can be used to treat autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, gastritis , Hashimoto's thyroiditis, ankylosing spondylitis, cholestyritis vasculitis associated with hepatitis C, chronic focal encephalitis, bullous pemphigoid, hemophilia A, membranous proliferative spheroid nephritis , granule syndrome, adult and adolescent dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, optic neuromyelitis, Gram's thyroid dysfunction Disease, bullous pemphigoid, membranous proliferative glomerulonephritis, Cheshire-Sydney syndrome and asthma. In another embodiment, the pharmaceutical composition is related to other conditions, such as transplant rejection, such as renal transplant rejection, liver transplant rejection, lung transplant rejection, cardiac transplant rejection, and bone marrow transplant rejection; graft resistance Host disease; viral infections such as mV infection, HIV infection, AIDS, etc.; and parasitic infections such as pear-shaped flagellosis, deformation Insects, schistosomiasis; and other conditions as determined by the general practitioner.

本發明提供醫藥組合物，其包含：a)有效量之本發明之抗體、抗體片段或抗體結合物；及b)醫藥學上可接受之載劑，其可為惰性或生理學活性的。 The invention provides a pharmaceutical composition comprising: a) an effective amount of an antibody, antibody fragment or antibody conjugate of the invention; and b) a pharmaceutically acceptable carrier which may be inert or physiologically active.

如本文所用之"醫藥學上可接受之載劑"包括生理學上相容之任何及所有溶劑、分散介質、包衣塗料、抗細菌及抗真菌劑及類似物。合適之載劑、稀釋劑及/或賦形劑之實例包括水、生理食鹽水、磷酸鹽緩衝生理食鹽水、右旋糖、丙三醇、乙醇及類似物中之一或多者及其組合。在許多狀況下，組合物中較佳包括諸如糖、多元醇或氯化鈉之等張劑。詳言之，合適載劑之相關實例包括：(1)含有或不含有約1mg/ml至25mg/ml人類血清白蛋白之杜貝卡氏(Dulbecco's)磷酸鹽緩衝生理食鹽水(pH值約7.4)，(2)0.9%生理食鹽水(0.9% w/v氯化鈉(NaCl))，及(3)5%(w/v)右旋糖；且亦可含有諸如色胺之抗氧化劑及諸如Tween 20之穩定劑。 "Pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents and/or excipients include one or more of water, physiological saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof . In many cases, it is preferred to include an isotonic agent such as a sugar, a polyol or sodium chloride in the composition. In particular, examples of suitable carriers include: (1) Dulbecco's phosphate buffered saline (with a pH of about 7.4) with or without about 1 mg/ml to 25 mg/ml human serum albumin. ), (2) 0.9% physiological saline (0.9% w/v sodium chloride (NaCl)), and (3) 5% (w/v) dextrose; and may also contain antioxidants such as tryptamine and Stabilizers such as Tween 20.

本文中之組合物亦可含有如接受治療之特定病症所必需之另一治療劑。較佳地，本發明之抗體、抗體片段或抗體結合物與補充活性化合物將具有不會彼此不利影響之互補活性。在一較佳實施例中，另一治療劑為表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、肝細胞生長因子(HGF)、組織因子(TF)、蛋白質C、蛋白質S、血小板衍生之生長因子(PDGF)、神經調節素-1(heregulin)、巨噬細胞刺激蛋白(MSP)或血管內皮生長因子(VEGF)之拮抗劑；或表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、肝細胞生長因子(HGF)、組織因子(TF)、蛋白質C、蛋白質S、血小板衍生之生長因子(PDGF)、神經調節素-1、巨噬細胞刺激蛋白(MSP)或血管內皮生長因子(VEGF)之受體(包括HER2受體、HER3受體、c-MET及其他受體酪胺酸激酶)之拮抗劑。在一較佳實施例中，另一治療劑為靶向分化群(CD)抗原之藥 劑，該等抗原包括CD3、CD14、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD36、CD40、CD44、CD52、CD55、CD59、CD56、CD70、CD79、CD80、CD103、CD134、CD137、CD138及CD152。在一較佳實施例中，另一治療劑為化學治療劑或免疫調節劑。 The compositions herein may also contain another therapeutic agent as is necessary for the particular condition being treated. Preferably, the antibodies, antibody fragments or antibody conjugates of the invention and the supplemental active compound will have complementary activities that do not adversely affect each other. In a preferred embodiment, the other therapeutic agent is epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelets. Derived growth factor (PDGF), neuromodulin-1 (heregulin), macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF) antagonist; or epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), neurotonin-1, macrophage stimulating protein (MSP) or vascular endothelium Antagonists of growth factor (VEGF) receptors, including the HER2 receptor, HER3 receptor, c-MET, and other receptor tyrosine kinases. In a preferred embodiment, the additional therapeutic agent is a drug that targets a differentiation group (CD) antigen Agents, such as CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138 and CD152. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic or immunomodulatory agent.

本發明之組合物可為多種形式。此等形式包括(例如)液體、半固體及固體劑型，但較佳形式視目標投藥模式及治療應用而定。典型較佳組合物為可注射或可輸注溶液之形式。較佳投藥模式為非經腸(例如靜脈內、肌肉內、腹膜內、皮下)。在一較佳實施例中，本發明之組合物係以快速注射形式或藉由經一段時間連續輸液來靜脈內投予。在另一較佳實施例中，其係藉由肌肉內、皮下、關節內、滑膜內、腫瘤內、腫瘤旁、病灶內或病灶旁途徑來注射以發揮局部以及全身治療作用。 The compositions of the present invention can take a wide variety of forms. Such forms include, for example, liquid, semi-solid, and solid dosage forms, but preferred forms will depend on the mode of administration and the therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions. The preferred mode of administration is parenteral (eg, intravenous, intramuscular, intraperitoneal, subcutaneous). In a preferred embodiment, the compositions of the invention are administered intravenously in a bolus injection or by continuous infusion over a period of time. In another preferred embodiment, it is administered by intramuscular, subcutaneous, intra-articular, intrasynovial, intratumoral, para-tumor, intralesional or paracancerous routes for local and systemic therapeutic effects.

用於非經腸投藥之無菌組合物可藉由將本發明之抗體、抗體片段或抗體結合物以所需量併入適當溶劑中，接著藉由微濾殺菌來製備。作為溶劑或媒劑，可使用水、生理食鹽水、磷酸鹽緩衝生理食鹽水、右旋糖、丙三醇、乙醇及類似物以及其組合。在許多狀況下，組合物中較佳包括諸如糖、多元醇或氯化鈉之等張劑。此等組合物亦可含有佐劑，尤其是濕潤劑、等張劑、乳化劑、分散劑及穩定劑。用於非經腸投藥之無菌組合物亦可以無菌固體組合物形式製備，可將該等無菌固體組合物在使用時溶解於無菌水或任何其他可注射無菌介質中。 Sterile compositions for parenteral administration can be prepared by incorporating the antibodies, antibody fragments or antibody conjugates of the invention in a suitable amount in a suitable solvent, followed by microfiltration sterilization. As the solvent or vehicle, water, physiological saline, phosphate buffered physiological saline, dextrose, glycerin, ethanol, and the like, and a combination thereof can be used. In many cases, it is preferred to include an isotonic agent such as a sugar, a polyol or sodium chloride in the composition. These compositions may also contain adjuvants, especially wetting agents, isotonic agents, emulsifying agents, dispersing agents and stabilizers. Sterile compositions for parenteral administration can also be prepared in the form of a sterile solid compositions which may be dissolved in sterile water or any other injectable sterile medium.

本發明之抗體、抗體片段或抗體結合物亦可經口投予。作為用於經口投藥之固體組合物，可使用錠劑、丸劑、散劑(明膠膠囊、藥囊)或顆粒。在此等組合物中，將本發明之活性成份與一或多種諸如澱粉、纖維素、蔗糖、乳糖或二氧化矽之惰性稀釋劑在氬氣流下混 合。此等組合物亦可包含除稀釋劑以外之物質，例如一或多種諸如硬脂酸鎂或滑石之潤滑劑、著色劑、包衣塗料(糖衣錠劑)或糖漿釉料。 The antibodies, antibody fragments or antibody conjugates of the invention may also be administered orally. As the solid composition for oral administration, a tablet, a pill, a powder (gelatin capsule, sachet) or granules can be used. In such compositions, the active ingredient of the present invention is mixed with one or more inert diluents such as starch, cellulose, sucrose, lactose or cerium oxide under an argon stream. Hehe. These compositions may also contain materials other than diluents, such as one or more lubricants such as magnesium stearate or talc, colorants, coatings (sugar coatings) or syrup glazes.

作為用於經口投藥之液體組合物，可使用含有諸如水、乙醇、丙三醇、植物油或石蠟油之惰性稀釋劑的醫藥學上可接受之溶液、懸浮液、乳液、糖漿及酏劑。此等組合物可包含除稀釋劑以外之物質，例如濕潤、增甜、增稠、調味或穩定化產品。 As the liquid composition for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing an inert diluent such as water, ethanol, glycerin, vegetable oil or paraffin oil can be used. Such compositions may contain materials other than diluents such as moisturizing, sweetening, thickening, flavoring or stabilizing products.

劑量視所需作用、治療持續時間及所用投藥途徑而定；對於成人而言，其一般介於每天口服5mg與1000mg之間，其中單位劑量在1mg至250mg活性物質之範圍內。一般而言，醫生將視待治療受檢者之特定年齡、體重及任何其他因素來確定適當劑量。 The dosage will depend on the desired effect, duration of treatment, and route of administration employed; for adults, it will generally be between 5 mg and 1000 mg orally per day, with unit doses ranging from 1 mg to 250 mg of active substance. In general, the physician will determine the appropriate dosage based on the particular age, weight and any other factors of the subject being treated.

治療使用方法Treatment use

在另一實施例中，本發明提供一種殺死CD38+細胞之方法，其係藉由將結合該CD38且能夠經由細胞凋亡、ADCC及/或CDC殺死該CD38+細胞之抗體投予有需要之患者。可治療性使用任何類型之本發明之抗體、抗體片段或細胞毒性結合物。因此，本發明包括抗-CD38單株抗體、其片段或其細胞毒性結合物作為藥劑之用途。 In another embodiment, the present invention provides a method of killing CD38 + cell method, by which the system and capable of binding the CD38 cells via apoptosis, the ADCC and / or CDC, cell killing of the CD38 + antibody administered to a subject Need patients. Any type of antibody, antibody fragment or cytotoxic conjugate of the invention can be used therapeutically. Thus, the invention encompasses the use of an anti-CD38 monoclonal antibody, a fragment thereof or a cytotoxic conjugate thereof as a medicament.

在一較佳實施例中，本發明之抗體、抗體片段或細胞毒性結合物係用於治療哺乳動物之過度增生性病症或發炎疾病或自體免疫疾病。在一更佳實施例中，將上文所揭示且含有本發明之抗體、抗體片段或細胞毒性結合物之醫藥組合物之一用於治療哺乳動物之過度增生性病症。在一實施例中，該病症為癌症。特定言之，該癌症為轉移性癌症。 In a preferred embodiment, the antibody, antibody fragment or cytotoxic conjugate of the invention is for use in the treatment of a hyperproliferative disorder or an inflammatory or autoimmune disorder in a mammal. In a more preferred embodiment, one of the pharmaceutical compositions disclosed above and comprising an antibody, antibody fragment or cytotoxic conjugate of the invention is used to treat a hyperproliferative disorder in a mammal. In one embodiment, the condition is cancer. In particular, the cancer is a metastatic cancer.

因此，本發明之醫藥組合物適用於治療或預防多種癌症，其包括(但不限於)以下各病症：癌瘤，包括膀胱、乳房、結腸、腎、肝、肺、卵巢、胰腺、胃、子宮頸、甲狀腺及皮膚之癌瘤；包括鱗狀細胞癌；淋巴系之造血腫瘤，包括白血病、急性淋巴細胞性白血病、急性 淋巴母細胞白血病、B細胞淋巴瘤、T細胞淋巴瘤、伯基特氏淋巴瘤；骨髓系之造血腫瘤，包括急性及慢性骨髓性白血病及前髓細胞性白血病；間葉源性腫瘤，包括纖維肉瘤及橫紋肌肉瘤；其他腫瘤，包括黑素瘤、精原細胞瘤、畸胎癌、神經母細胞瘤及神經膠質瘤；中樞及周邊神經系統之腫瘤，包括星形細胞瘤、神經母細胞瘤、神經膠質瘤及神經鞘瘤；間葉源性腫瘤，包括纖維肉瘤、橫紋肌肉瘤及骨肉瘤；及其他腫瘤，包括黑素瘤、著色性乾皮病、角化棘皮瘤、精原細胞瘤、甲狀腺濾泡癌及畸胎癌；及仍待確定之其中表現CD38之其他癌症。較佳地，該病症為其中表現CD38之NHL、BL、MM、B-CLL、ALL、TCL、AML、HCL、HL或CML，及仍待確定之其中CD38優勢表現之其他癌症。在另一實施例中，本發明之醫藥組合物可用於治療自體免疫疾病，諸如全身性紅斑性狼瘡症、類風濕性關節炎、多發性硬化症、克隆氏病、潰瘍性結腸炎、胃炎、橋本氏甲狀腺炎、強直性脊椎炎、C型肝炎相關之冷凝球蛋白性血管炎、慢性局竈性部腦炎、大疱性類天疱瘡、A型血友病、膜性增生性絲球體腎炎、修格蘭氏症候群、成人及青少年皮肌炎、成人多發性肌炎、慢性蕁麻疹、原發性膽汁性肝硬化症、特發性血小板減少性紫癜、視神經脊髓炎、格雷氏甲狀腺機能障礙疾病、大疱性類天疱瘡、膜性增生性絲球體腎炎、徹奇-斯全司症候群及哮喘。在另一實施例中，該醫藥組合物係關於其他病症，諸如移植排斥反應，諸如腎移植排斥反應、肝移植排斥反應、肺移植排斥反應、心臟移植排斥反應及骨髓移植排斥反應；移植物抗宿主疾病；病毒感染，諸如mV感染、HIV感染、AIDS等；及寄生蟲感染，諸如梨形鞭毛蟲症、變形蟲症、血吸蟲病；及如由一般技術者所確定之其他病症。 Accordingly, the pharmaceutical composition of the present invention is useful for treating or preventing a variety of cancers including, but not limited to, the following disorders: cancer, including bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, son Carcinoma of the cervix, thyroid, and skin; including squamous cell carcinoma; hematopoietic tumors of the lymphoid system, including leukemia, acute lymphocytic leukemia, acute Lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of the myeloid lineage, including acute and chronic myeloid leukemia and promyelocytic leukemia; mesenchymal tumors, including fibers Sarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, Glioma and schwannomas; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid Follicular cancer and teratocarcinoma; and other cancers that still have to be identified that exhibit CD38. Preferably, the condition is NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL or CML in which CD38 is expressed, and other cancers in which the CD38 dominant performance is still to be determined. In another embodiment, the pharmaceutical composition of the present invention can be used to treat autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, gastritis , Hashimoto's thyroiditis, ankylosing spondylitis, cholestyritis vasculitis associated with hepatitis C, chronic focal encephalitis, bullous pemphigoid, hemophilia A, membranous hypertrophic spheroid Nephritis, repairing Gram syndrome, adult and adolescent dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, optic neuromyelitis, Gram's thyroid function Obstructive diseases, bullous pemphigoid, membranous proliferative spheroid nephritis, Church-Scott syndrome and asthma. In another embodiment, the pharmaceutical composition is related to other conditions, such as transplant rejection, such as renal transplant rejection, liver transplant rejection, lung transplant rejection, cardiac transplant rejection, and bone marrow transplant rejection; graft resistance Host disease; viral infections, such as mV infection, HIV infection, AIDS, etc.; and parasitic infections, such as pear-shaped flagellate, amoeba, schistosomiasis; and other conditions as determined by the ordinarily skilled artisan.

類似地，本發明提供一種用於抑制所選細胞群體之生長之方法，其包含使靶細胞或含有靶細胞之組織與有效量之單獨或與其他細 胞毒性劑或治療劑組合之本發明之抗體、抗體片段或抗體結合物，或構成細胞毒性結合物之抗體、抗體片段或治療劑接觸。在一較佳實施例中，另一治療劑為表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、肝細胞生長因子(HGF)、組織因子(TF)、蛋白質C、蛋白質S、血小板衍生之生長因子(PDGF)、神經調節素-1、巨噬細胞刺激蛋白(MSP)或血管內皮生長因子(VEGF)之拮抗劑；或表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、肝細胞生長因子(HGF)、組織因子(TF)、蛋白質C、蛋白質S、血小板衍生之生長因子(PDGF)、神經調節素-1、巨噬細胞刺激蛋白(MSP)或血管內皮生長因子(VEGF)之受體(包括HER2受體、HER3受體、c-MET及其他受體酪胺酸激酶)之拮抗劑。在一較佳實施例中，另一治療劑為靶向分化群(CD)抗原之藥劑，該等抗原包括CD3、CD14、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD36、CD40、CD44、CD52、CD55、CD59、CD56、CD70、CD79、CD80、CD103、CD134、CD137、CD138及CD152。在一較佳實施例中，另一治療劑為化學治療劑或免疫調節劑。 Similarly, the present invention provides a method for inhibiting growth of a selected population of cells comprising or consisting of a target cell or a tissue containing the target cell and an effective amount alone or in combination with other An antibody, antibody fragment or antibody conjugate of the invention, or an antibody, antibody fragment or therapeutic agent comprising a cytotoxic conjugate, in combination with a cytotoxic agent or combination of therapeutic agents. In a preferred embodiment, the other therapeutic agent is epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelets. Derived growth factor (PDGF), neuromodulin-1, macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF) antagonist; or epidermal growth factor (EGF), fibroblast growth factor (FGF) , hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), neurotonin-1, macrophage stimulating protein (MSP) or vascular endothelial growth factor ( Antagonists of VEGF) receptors, including the HER2 receptor, HER3 receptor, c-MET, and other receptor tyrosine kinases. In a preferred embodiment, the other therapeutic agent is an agent that targets a differentiation group (CD) antigen, including CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138 and CD152. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic or immunomodulatory agent.

用於抑制所選細胞群體之生長之方法可活體外、活體內或離體實施。如本文所用之"抑制生長"意謂歷經短時段或長時段的減緩細胞生長、降低細胞生存力、致使細胞死亡、溶解細胞及誘發細胞死亡。 Methods for inhibiting growth of a selected population of cells can be performed in vitro, in vivo or ex vivo. "Inhibiting growth" as used herein means slowing cell growth, reducing cell viability, causing cell death, lysing cells, and inducing cell death over a short period of time or for a prolonged period of time.

活體外用途之實例包括在自體骨髓移植至同一患者中之前處理自體骨髓以殺死病變或惡性細胞；在骨髓移植前處理骨髓以殺死感受態T細胞及預防移植物抗宿主疾病(GVHD)；處理細胞培養物以殺死除不表現靶抗原之所要變異體以外之所有細胞或殺死表現不當抗原之變異體。 Examples of in vitro use include treating autologous bone marrow to kill diseased or malignant cells prior to autologous bone marrow transplantation into the same patient; treating bone marrow prior to bone marrow transplantation to kill competent T cells and preventing graft versus host disease (GVHD) The cell culture is treated to kill all cells other than the desired variant that does not represent the target antigen or to kill variants that exhibit inappropriate antigens.

非臨床活體外使用之條件易於由一般技術者確定。 The conditions for non-clinical in vitro use are readily determined by the average skilled artisan.

臨床離體用途之實例為在癌症治療中或在自體免疫疾病治療中 在自體移植前自骨髓移除腫瘤細胞或淋巴細胞，或在移植前自自體或同種異體骨髓或組織移除T細胞及其他淋巴細胞以預防移植物抗宿主疾病(GVHD)。治療可如下進行。自患者或其他個體採集骨髓且接著在含有其中添加本發明之細胞毒性劑之血清的培養基中培育。濃度在約10μM至1pM之範圍內，在約37℃下歷時約30分鐘至約48小時。培育濃度及時間之確切條件(亦即劑量)易於由一般技術者確定。在培育後，將骨髓細胞用含有血清之培養基洗滌且藉由根據已知方法靜脈內輸液而返回患者體內。在患者於採集骨髓與回輸經治療細胞之時間之間接受諸如去除式(ablative)化學療法或全身照射之療程的其他治療之情形中，使用標準醫用設備將經治療骨髓細胞冷凍儲存於液氮中。 Examples of clinical ex vivo use are in the treatment of cancer or in the treatment of autoimmune diseases Tumor cells or lymphocytes are removed from the bone marrow prior to autologous transplantation, or T cells and other lymphocytes are removed from autologous or allogeneic bone marrow or tissue prior to transplantation to prevent graft versus host disease (GVHD). Treatment can be carried out as follows. Bone marrow is collected from a patient or other individual and then incubated in a medium containing serum to which the cytotoxic agent of the present invention is added. The concentration is in the range of from about 10 [mu]M to 1 pM at about 37[deg.] C. for about 30 minutes to about 48 hours. The exact conditions (i.e., dosage) at which the concentration and time are cultivated are readily determined by the average skilled artisan. After incubation, the bone marrow cells are washed with serum-containing medium and returned to the patient by intravenous infusion according to known methods. In the case where the patient receives other treatments such as ablative chemotherapy or whole body irradiation between the time of collecting the bone marrow and returning the treated cells, the treated bone marrow cells are stored frozen in liquid using standard medical equipment. In nitrogen.

對於臨床活體內使用而言，本發明之抗體、抗原決定基結合抗體片段或細胞毒性結合物將以經測試無菌性及內毒素含量之溶液之形式供給。細胞毒性結合物投藥之合適方案之實例如下。結合物每週給予一次歷時4週，每週以靜脈內快速注射之形式給予。單次劑量係在其中可添加5至10ml人類血清白蛋白之50至100ml生理食鹽水中給予。每次靜脈內投藥劑量可為10μg至100mg(每天在100ng至1mg/kg之範圍內)。更佳地，劑量將在50μg至30mg之範圍內。最佳地，劑量將在1mg至20mg之範圍內。治療四週後，患者可基於每週一次繼續接受治療。關於投藥途徑、賦形劑、稀釋劑、劑量、時間等之特定臨床方案可由一般技術者根據臨床情況來確定。 For clinical in vivo use, the antibodies, epitope-binding antibody fragments or cytotoxic conjugates of the invention will be supplied as a solution of the tested sterility and endotoxin content. Examples of suitable protocols for administration of cytotoxic conjugates are as follows. The conjugate was administered once a week for 4 weeks and was administered weekly as a rapid intravenous injection. A single dose is administered in 50 to 100 ml of physiological saline to which 5 to 10 ml of human serum albumin can be added. The dose may be from 10 μg to 100 mg per dose (in the range of 100 ng to 1 mg/kg per day). More preferably, the dosage will be in the range of 50 μg to 30 mg. Most preferably, the dosage will be in the range of 1 mg to 20 mg. After four weeks of treatment, the patient can continue to receive treatment on a weekly basis. Specific clinical regimens regarding routes of administration, excipients, diluents, dosages, times, etc., can be determined by one of ordinary skill in the light of the clinical circumstances.

診斷diagnosis

本發明之抗體或抗體片段亦可用於活體外或活體內偵側生物樣本中之CD38。在一實施例中，本發明之抗-CD38抗體係用於測定組織中或來源於該組織之細胞中之CD38含量。在一較佳實施例中，該組織為病變組織。在該方法之一較佳實施例中，組織為腫瘤或其活組織檢查切片。在該方法之一較佳實施例中，首先自患者切除組織或其活 組織檢查切片，且接著可在免疫檢定中以本發明之抗體或抗體片段測定組織或活組織檢查切片中之CD38含量。在另一較佳實施例中，CD38含量係針對可經冷凍或固定之組織或其活組織檢查切片之樣本來測定。相同方法可用於測定CD38蛋白之其他特性，諸如其細胞表面含量或其細胞定位。 The antibody or antibody fragment of the invention can also be used for CD38 in a biological sample in vitro or in vivo. In one embodiment, the anti-CD38 anti-system of the invention is used to determine the amount of CD38 in a tissue or cells derived from the tissue. In a preferred embodiment, the tissue is a diseased tissue. In a preferred embodiment of the method, the tissue is a tumor or a biopsy section thereof. In a preferred embodiment of the method, the tissue is first removed from the patient or lived The sections are examined by tissue and the CD38 content in the tissue or biopsy sections can then be determined in an immunoassay using the antibodies or antibody fragments of the invention. In another preferred embodiment, the CD38 content is determined for a sample that can be frozen or fixed tissue or a biopsy slice thereof. The same method can be used to determine other properties of the CD38 protein, such as its cell surface content or its cellular localization.

上述方法可用於診斷已知或懷疑患有癌症之受檢者之癌症，其中將在該患者中所量測之CD38含量與正常參考受檢者或標準之CD38含量比較。該方法可接著用於判定腫瘤是否表現CD38，腫瘤表現CD38可表明該腫瘤將良好回應於本發明之抗體、抗體片段或抗體結合物之治療。較佳地，腫瘤為其中表現CD38之NHL、BL、MM、B-CLL、ALL、TCL、AML、HCL、HL或CML，及仍待確定之其中CD38優勢表現之其他癌症。 The above method can be used to diagnose cancer in a subject known or suspected of having cancer, wherein the CD38 content measured in the patient is compared to the normal reference subject or standard CD38 content. This method can then be used to determine whether a tumor exhibits CD38, and a tumor exhibiting CD38 can indicate that the tumor will respond well to treatment with an antibody, antibody fragment or antibody conjugate of the invention. Preferably, the tumor is an NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL or CML in which CD38 is expressed, and other cancers in which the CD38 dominant performance is still to be determined.

本發明進一步提供經另外標記以用於研究或診斷應用之單株抗體、人類化抗體及其抗原決定基結合片段。在較佳實施例中，標記為放射性標記、螢光團、發色團、成像劑或金屬離子。 The invention further provides monoclonal antibodies, humanized antibodies, and epitope binding fragments thereof that are additionally labeled for use in research or diagnostic applications. In a preferred embodiment, the label is a radioactive label, a fluorophore, a chromophore, an imaging agent, or a metal ion.

亦提供一種診斷方法，其中將該等經標記之抗體或其抗原決定基結合片段投予疑患癌症或發炎疾病或自體免疫疾病之受檢者，且量測或監測該受檢者體內之標記分布。 Also provided is a diagnostic method wherein the labeled antibody or its epitope binding fragment is administered to a subject suspected of having cancer or an inflammatory disease or an autoimmune disease, and measuring or monitoring the subject Tag distribution.

套組Set

本發明亦包括(例如)包含所述細胞毒性結合物及該細胞毒性結合物用於殺死特定細胞類型之使用說明書的套組。該等說明書可包括活體外、活體內或離體使用細胞毒性結合物之指導。 The invention also includes, for example, a kit comprising the cytotoxic conjugate and instructions for use of the cytotoxic conjugate to kill a particular cell type. Such instructions may include instructions for the use of cytotoxic conjugates in vitro, in vivo or ex vivo.

通常，套組將具有含有細胞毒性結合物之隔區。細胞毒性結合物可為凍乾形式、液體形式或可經改適以包括於套組中之其他形式。套組亦可含有實施套組中之說明書上所述之方法所需之其他要素，諸如用於使凍乾粉末復水之經殺菌溶液、用於在投予患者前與細胞毒性 結合物組合之其他藥劑及幫助將結合物投予患者之工具。 Typically, the kit will have a compartment containing a cytotoxic conjugate. The cytotoxic combination can be in lyophilized form, in liquid form, or in other forms that can be adapted to be included in the kit. The kit may also contain other elements required to carry out the methods described in the instructions in the kit, such as a sterilizing solution for reconstituting the lyophilized powder, for administration to a patient prior to administration to cytotoxicity Other agents of the combination of conjugates and tools that aid in administering the conjugate to the patient.

實例Instance

現參考以下實例描述本發明，該等實例僅為說明性的且並不意欲限制本發明。 The invention is described with reference to the following examples, which are merely illustrative and not intended to limit the invention.

實例1Example 1 小鼠CD38抗體Mouse CD38 antibody

使用穩定表現高含量人類CD38之300-19細胞(一種來源於Balb/c小鼠之前B細胞株)(M.G.Reth等人1985,Nature,317：353-355)免疫Balb/c VAF小鼠。藉由在ImmunoGen,Inc.使用標準免疫方案以每2-3週每隻小鼠約5×106個表現CD38之300-19細胞經皮下免疫小鼠。在處死以產生融合瘤之前三天用另一劑量之抗原補強經免疫小鼠。根據標準動物方案採集小鼠之脾且將其在兩個無菌、磨砂載玻片之間研磨以獲得於RPMI-1640培養基中之單細胞懸浮液。將脾細胞粒化，洗滌，且藉由使用聚乙二醇-1500(Roche 783 641)使其與鼠科骨髓瘤P3X63Ag8.653細胞融合(J.F.Kearney等人1979,J Immunol,123：1548-1550)。將融合細胞再懸浮於含有次黃嘌呤-胺基喋呤-胸苷(HAT)(Sigma H-0262)之RPMI-1640選擇培養基中且經選擇以在37℃(5% CO2)下在96孔平底培養盤(Corning-Costar 3596，每孔200μL細胞懸浮液)中生長。培育5天後，自各孔移除100μL培養上清液且用100μL含有次黃嘌呤-胸苷(HT)補充物(Sigma H-0137)之RPMI-1640培養基替換。在37℃(5% CO2)下繼續培育直至融合瘤純系備妥用於抗體篩選。亦可使用免疫及融合瘤產生之其他技術，包括J.Langone及H.Vunakis(編，Methods in Enzymology，第121卷，"Immunochemical Techniques,Part I"；Academic Press,Florida)及E.Harlow及D.Lane("Antibodies：A Laboratory Manual"；1988；Cold Spring Harbor Laboratory Press,New York)所述之技術。 Balb/c VAF mice were immunized with 300-19 cells (a B cell strain derived from Balb/c mice) stably expressing high levels of human CD38 (MG Reth et al. 1985, Nature , 317: 353-355). With the ImmunoGen, Inc. Using standard immunization protocols per mouse every 2-3 weeks to approximately 5 × 10 6 th CD38 expression 300-19 cells of mice immunized subcutaneously. The immunized mice were reinforced with another dose of antigen three days prior to sacrifice to produce a fusion tumor. The spleens of mice were harvested according to standard animal protocols and ground between two sterile, frosted slides to obtain a single cell suspension in RPMI-1640 medium. Spleen cells were granulated, washed, and fused with murine myeloma P3X63Ag8.653 cells by using polyethylene glycol-1500 (Roche 783 641) (JF Kearney et al. 1979, J Immunol , 123: 1548-1550) . The fused cells were resuspended in RPMI-1640 selection medium containing hypoxanthine-aminopurine-thymidine (HAT) (Sigma H-0262) and selected to be at 37 ° C (5% CO 2 ) at 96 A well flat bottom plate (Corning-Costar 3596, 200 [mu]L cell suspension per well) was grown. After 5 days of incubation, 100 μL of the culture supernatant was removed from each well and replaced with 100 μL of RPMI-1640 medium containing hypoxanthine-thymidine (HT) supplement (Sigma H-0137). Incubation was continued at 37 ° C (5% CO 2 ) until the fusion tumor was prepared for antibody screening. Other techniques for immunization and fusion tumors can also be used, including J. Langone and H. Vunakis (eds., Methods in Enzymology , Vol. 121, "Immunochemical Techniques, Part I"; Academic Press, Florida) and E. Harlow and D. .Lane ("Antibodies: A Laboratory Manual";1988; Cold Spring Harbor Laboratory Press, New York).

藉由使用Becton Dickinson FACSCalibur或FACSArray機器進行螢光活化細胞分選(FACS)，對來自融合瘤之培養上清液針對分泌與表現CD38之300-19細胞結合，但不與母體300-19細胞結合之小鼠單株抗體進行篩選(使用結合FITC或PE之抗小鼠IgG抗血清)。次選殖測試為陽性之融合瘤純系，且使用商業分型試劑(Roche 1493027)鑑別各經分泌抗-CD38抗體之同型。將對於CD38結合呈陽性之總共29種抗體藉由使用標準方案之蛋白質A或G層析來純化且接著進一步表徵。 By using a Becton Dickinson FACSCalibur or FACSArray machine for fluorescence activated cell sorting (FACS), culture supernatants from fusion tumors were secreted against 300-19 cells secreting and expressing CD38, but not bound to maternal 300-19 cells. Mouse monoclonal antibodies were screened (using anti-mouse IgG antiserum combined with FITC or PE). The subclonal test was positive for the fusion tumor, and the commercial isotyping reagent (Roche 1493027) was used to identify the isotype of each secreted anti-CD38 antibody. A total of 29 antibodies positive for CD38 binding were purified by protein A or G chromatography using standard protocols and then further characterized.

實例2Example 2 抗-CD38抗體之結合表徵Binding characterization of anti-CD38 antibodies

證明抗-CD38抗體38SB13、38SB18、38SB19、38SB30、38SB31及38SB39與表現CD38之300-19細胞結合且不存在與母體300-19細胞結合之FACS直方圖展示於圖1中。將38SB13、38SB18、38SB19、38SB30、38SB31或38SB39抗體(10nM)與表現CD38之300-19細胞或母體300-19細胞(每個樣本1-2×105個細胞)在100μL補充2%正常山羊血清之冰冷RPMI-1640培養基中一起培育3h。接著，將細胞粒化，洗滌，且在冰上與結合FITC之山羊抗小鼠IgG抗體(Jackson Laboratory，100μL，6μg/mL於補充2%正常山羊血清之冷RPMI-1640培養基中)一起培育1h。將細胞再次粒化，洗滌，再懸浮於200μL含有1%甲醛之PBS中，且使用FACSCalibur流式細胞儀以CellQuest軟體(BD Biosciences)進行分析。 FACS histograms demonstrating that anti-CD38 antibodies 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 bind to 300-19 cells expressing CD38 and are absent from maternal 300-19 cells are shown in Figure 1. 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibody (10nM) with 300-19 cells expressing CD38 or maternal 300-19 cells (1-2×10 5 cells per sample) supplemented with 2% normal goats at 100 μL Serum was incubated in ice-cold RPMI-1640 medium for 3 h. Next, the cells were granulated, washed, and incubated with FITC-conjugated goat anti-mouse IgG antibody (Jackson Laboratory, 100 μL, 6 μg/mL in cold RPMI-1640 medium supplemented with 2% normal goat serum) for 1 h on ice. . The cells were re-granulated, washed, resuspended in 200 μL of PBS containing 1% formaldehyde, and analyzed by CellQuest software (BD Biosciences) using a FACSCalibur flow cytometer.

與相應陰性對照組(僅與結合FITC之山羊抗小鼠IgG抗體一起培育之細胞)相比，與38SB13、38SB18、38SB19、38SB30、38SB31或38SB39一起培育之表現CD38之300-19細胞之FACS直方圖展示出強螢光位移(圖1)。另外，當將母體300-19細胞與此等抗體中之任一者一起培育時，未偵側到顯著螢光位移。當使用陽性對照抗-CD38抗體AT13/5(Serotec，MCA1019)時獲得類似結果。 FACS histogram of 300-19 cells expressing CD38 incubated with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 compared to the corresponding negative control group (cells incubated with FITC-conjugated goat anti-mouse IgG antibody) The figure shows a strong fluorescence shift (Figure 1). In addition, when the parental 300-19 cells were incubated with any of these antibodies, no significant fluorescence shift was detected. Similar results were obtained when the positive control anti-CD38 antibody AT13/5 (Serotec, MCA1019) was used.

當將Ramos(ATCC CRL 1596)淋巴瘤細胞與38SB13、38SB18、38SB19、38SB30、38SB31或38SB39一起培育時亦觀測到強螢光位移(圖1)。使用針對S形劑量反應曲線之非線性回歸方法(GraphPad Prizm，第4版，軟體，San Diego,CA)，自圖2所示之FACS分析曲線評估38SB13、38SB18、38SB19、38SB30、38SB31及38SB39與Ramos細胞結合之表觀解離常數(KD)之值。該等值如下：分別為0.10nM、0.10nM、0.12nM、0.16nM、0.11nM及3.03nM。 Strong fluorescence shifts were also observed when Ramos (ATCC CRL 1596) lymphoma cells were incubated with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 (Figure 1). Evaluation of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 from the FACS analysis curve shown in Figure 2 using a non-linear regression method for sigmoidal dose response curves (GraphPad Prizm, 4th edition, software, San Diego, CA) The value of the apparent dissociation constant (K D ) of Ramos cell binding. The values are as follows: 0.10 nM, 0.10 nM, 0.12 nM, 0.16 nM, 0.11 nM, and 3.03 nM, respectively.

實例3 Example 3 38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗體對Ramos及Daudi淋巴瘤細胞凋亡之誘發Induction of Apoptosis of Ramos and Daudi Lymphoma Cells by 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 Antibodies

抗-CD38抗體38SB13、38SB18、38SB19、38SB30、38SB31及38SB39誘發Ramos及Daudi(ATCC CCL-213)淋巴瘤細胞株及MOLP-8多發性骨髓瘤細胞株(DSMZ ACC 569)凋亡。在用Annexin V之FITC結合物(Biosource PHN1018)及用TO-PRO-3(Invitrogen T3605)染色後藉由FACS分析量測細胞凋亡程度。Annexin V結合完整細胞之細胞膜雙層之外的磷脂醯絲胺酸而不結合其內之磷脂醯絲胺酸。在健康、正常細胞中，磷脂醯絲胺酸係在膜雙層之內表現，且磷脂醯絲胺酸自質膜內層轉移至質膜外層為細胞凋亡之可最早可偵側信號之一。因此，Annexin V結合為誘發細胞凋亡之信號。TO-PRO-3為當如細胞凋亡之後期階段所發生之膜完整性破壞時可僅穿透質膜之單體花青核酸染料。 Anti-CD38 antibodies 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 induced apoptosis in Ramos and Daudi (ATCC CCL-213) lymphoma cell lines and MOLP-8 multiple myeloma cell line (DSMZ ACC 569). The degree of apoptosis was measured by FACS analysis after staining with FITC conjugate of Annexin V (Biosource PHN1018) and with TO-PRO-3 (Invitrogen T3605). Annexin V binds phospholipid ceramide outside the cell membrane bilayer of intact cells without binding to phospholipid lysine in it. In healthy and normal cells, phospholipid yrosine acid is expressed in the membrane bilayer, and phospholipid lysine is transferred from the inner layer of the plasma membrane to the outer layer of the plasma membrane, which is one of the earliest detectable signals. . Therefore, Annexin V binds to a signal that induces apoptosis. TO-PRO-3 is a monomeric cyanine nucleic acid dye that can only penetrate the plasma membrane when membrane integrity is disrupted, such as occurs in the later stages of apoptosis.

將指數生長細胞以約2×105個細胞/毫升塗於24孔盤中之補充10%胎牛血清(FBS)、2mM L-麩胺醯胺及50μg/mL慶大黴素(gentamycin)之RMPI-1640培養基(下文表示為完全RMPI-1640培養基)中。除非另有規定，否則細胞一般係在完全RMPI-1640培養基中生長。在37℃下在含有5% CO2之潮濕氣氛中將細胞與抗-CD38抗體(10nM)一起培育 24h。接著將細胞粒化，用500μL PBS洗滌兩次，再懸浮於100μL含有5μL Annexin V-FITC之結合緩衝液(Annexin V-FITC套組中提供)中，且在冰上培育15min。接著，將400μL結合緩衝液及TO-PRO-3(達到1μM之最終濃度)添加至混合物中，且藉由FACS立即量測FITC及TO-PRO-3之細胞結合螢光。對於各樣本收集4000個事件。使用CellQuest軟體產生TO-PRO-3之螢光(FL4-H；y軸)及Annexin V-FITC之螢光(FL1-H；x軸)之點陣圖。 The exponentially growing cells were applied to a 24-well plate at a dose of about 2×10 5 cells/ml to supplement 10% fetal calf serum (FBS), 2 mM L-glutamine and 50 μg/mL gentamycin. RMPI-1640 medium (hereinafter referred to as complete RMPI-1640 medium). Unless otherwise specified, cells are typically grown in complete RMPI-1640 medium. Cells were incubated with anti-CD38 antibody (10 nM) for 24 h at 37 ° C in a humidified atmosphere containing 5% CO 2 . The cells were then granulated, washed twice with 500 μL of PBS, resuspended in 100 μL of binding buffer containing 5 μL of Annexin V-FITC (provided in the Annexin V-FITC kit), and incubated on ice for 15 min. Next, 400 μL of binding buffer and TO-PRO-3 (to a final concentration of 1 μM) were added to the mixture, and cell-bound fluorescence of FITC and TO-PRO-3 was immediately measured by FACS. 4000 events were collected for each sample. A dot plot of TO-PRO-3 fluorescence (FL4-H; y-axis) and Annexin V-FITC fluorescence (FL1-H; x-axis) was generated using CellQuest software.

結果展示於圖3及圖4中。圖3給出Daudi細胞在與各種抗-CD38抗體一起培育24h後之該點陣圖之一實例。自此等圖示確定來自雙重複樣本之Annexin V陽性細胞(包括TO-PRO-3陽性與陰性細胞)之平均百分數且展示於圖4中。意外發現38SB13、38SB18、38SB19、38SB30、38SB31及38SB39顯示強細胞凋亡活性。與僅約6%之未經處理細胞相比，大於30%之暴露於此等抗體中之任一者之Daudi細胞呈Annexin V陽性(圖3及圖4A)。38SB13、38SB18、38SB19、38SB30、38SB31及38SB39顯示出比在10nM之相同濃度下測試之先前技術鼠科CD38抗體(AT13/5、OKT10、IB4及SUN-4B7，在減去未經處理對照值後小於10% Annexin V陽性)及在吾人實驗室中產生之兩種其他抗-CD38抗體(38SB7及38SB23，不高於未經處理對照組，亦即約6% Annexin V陽性)強至少2.4倍之細胞凋亡活性(在減去未經處理對照值後為24%)(圖4A)。AT13/5係購自Serotec(MCA1019)且OKT10係自融合瘤(ATCC CRL-8022)產生及純化。IB4及SUN-4B7為來自Prof.F.Malavasi,University,Turin,Italy之贈品。類似地，38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗-CD38抗體展示出比先前技術鼠科CD38抗體AT13/5、OKT10、IB4及SUN-4B7或兩種其他新抗-CD38抗體38SB7及38SB23(在減去未經處理對照值後小於2% Annexin V陽性)強至少3.5倍之對另一淋巴瘤細胞株Ramos之促細胞凋 亡活性倍(在減去非經處理對照值後7%或更多Annexin-V陽性)(圖4B)。最後，38SB13、38SB18、38SB19、38SB30、38SB31及38SB39抗-CD38抗體展示出對多發性骨髓瘤細胞株MOLP-8之強促細胞凋亡活性(圖4C)。與約39%之未經處理細胞相比，約50%之經此等抗體處理之MOLP-8細胞呈Annexin V陽性。相比之下，經先前技術鼠科CD38抗體AT13/5、OKT10、IB4及SUN-4B7或兩種其他新抗-CD38抗體38SB7及38SB23中之任一者處理並不使凋亡細胞部分增加。 The results are shown in Figures 3 and 4. Figure 3 shows an example of this dot pattern of Daudi cells after 24 h incubation with various anti-CD38 antibodies. The average percentage of Annexin V positive cells (including TO-PRO-3 positive and negative cells) from the double replicate samples was determined from these graphs and is shown in Figure 4. It was unexpectedly found that 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 showed strong apoptotic activity. More than 30% of Daudi cells exposed to any of these antibodies were Annexin V positive compared to only about 6% of untreated cells (Figure 3 and Figure 4A). 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 showed prior art murine CD38 antibodies (AT13/5, OKT10, IB4, and SUN-4B7, tested at the same concentration of 10 nM after subtracting untreated control values) Less than 10% Annexin V positive) and two other anti-CD38 antibodies produced in our laboratory (38SB7 and 38SB23, no higher than the untreated control, ie about 6% Annexin V positive) are at least 2.4 times stronger Apoptotic activity (24% after subtraction of untreated control values) (Fig. 4A). AT13/5 was purchased from Serotec (MCA1019) and the OKT10 line was produced and purified from a fusion tumor (ATCC CRL-8022). IB4 and SUN-4B7 are gifts from Prof. F. Malavasi, University, Turin, Italy. Similarly, the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 anti-CD38 antibodies exhibited AT13/5, OKT10, IB4 and SUN-4B7 or two other new anti-CD38 antibodies 38SB7 and 38SB23 compared to the prior art murine CD38 antibodies. (less than 2% Annexin V positive after subtracting the untreated control value) is at least 3.5 times stronger than the other lymphoma cell line Ramos Death activity (7% or more Annexin-V positive after subtracting the non-treated control value) (Fig. 4B). Finally, the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 anti-CD38 antibodies exhibited strong pro-apoptotic activity against the multiple myeloma cell line MOLP-8 (Fig. 4C). About 50% of the MOLP-8 cells treated with these antibodies were positive for Annexin V compared to about 39% of the untreated cells. In contrast, treatment with either the prior art murine CD38 antibodies AT13/5, OKT10, IB4 and SUN-4B7 or two other new anti-CD38 antibodies 38SB7 and 38SB23 did not partially increase apoptotic cells.

實例4Example 4 抗-CD38抗體之輕鏈及重鏈之選殖及定序Selection and sequencing of light and heavy chains of anti-CD38 antibodies 38SB19抗體38SB19 antibody 自產生38SB19抗體之融合瘤細胞製備RNA Preparation of RNA from fusion tumor cells producing 38SB19 antibody

使用Qiagen's RNeasy小規模純化套組，自產生38SB19抗體之5×106個融合瘤細胞獲得總RNA製劑。簡言之，將5×106個細胞粒化且再懸浮於350μL RLT緩衝液(含有1% β-巰基乙醇)中。藉由使懸浮液穿過21.5號針及注射器約10-20次或直至其不再具黏性來將其均質化。將乙醇(350μL之70%含水乙醇)添加至均質物中，將其充分混合。將溶液轉移至離心管柱，將管柱置放於2mL收集管中且以大於8000 x g旋轉15秒鐘。將管柱用500μL RPE緩衝液洗滌兩次，接著轉移至新鮮管中且用30μL無RNase水溶離且旋轉1分鐘。將溶離液(30μL)返回置於管柱上以進行第二次1分鐘之溶離旋轉。將30μL溶離液之等分試樣用水稀釋且用於量測260nm下之UV吸收來定量RNA。 Using Qiagen's RNeasy kit small-scale purification, 38SB19 antibody produced from 5 × 10 6 tumor cells obtained integrates both total RNA preparation. Briefly, 5 x 10 6 cells were pelleted and resuspended in 350 μL of RLT buffer (containing 1% β-mercaptoethanol). The suspension is homogenized by passing the suspension through a 21.5 gauge needle and syringe about 10-20 times or until it is no longer tacky. Ethanol (350 μL of 70% aqueous ethanol) was added to the homogenate and thoroughly mixed. The solution was transferred to a centrifuge column, which was placed in a 2 mL collection tube and spun at greater than 8000 xg for 15 seconds. The column was washed twice with 500 μL of RPE buffer, then transferred to a fresh tube and lysed with 30 μL of RNase-free water and spun for 1 minute. The eluate (30 [mu]L) was returned to the column for a second 1 minute dissolution spin. An aliquot of 30 μL of the eluate was diluted with water and used to measure UV absorbance at 260 nm to quantify RNA.

以逆轉錄酶(RT)反應製備cDNA Preparation of cDNA by reverse transcriptase (RT) reaction

使用Invitrogen之SuperscriptII套組自總RNA產生可變區38SB19抗體cDNA。嚴格遵循套組方案，利用來自Qianeasy小規模純化之多達5μg之總RNA。簡言之，用無RNase無菌蒸餾水使RNA、1μL隨機引子及1μL dNTP混合物達到12μL且在65℃下培育5分鐘。接著將混合物 置於冰上歷時至少1分鐘。接著添加4μL之5×反應緩衝液、2μL 0.1M DTT及1μL RNaseOUT且在25℃下將混合物在MJ Research熱循環儀中培育2分鐘。將熱循環儀暫停以便添加1μL SuperscriptII酶且接著重新啟動在25℃下再進行10分鐘，隨後轉換至55℃歷時50分鐘。藉由加熱至70℃歷時15min將反應熱滅活且藉由添加1μL RNase H且在37℃下培育20分鐘來移除RNA。 Variable region 38SB19 antibody cDNA was generated from total RNA using Invitrogen's Superscript II kit. Strictly follow the kit protocol, using up to 5 μg of total RNA from Qianeasy's small scale purification. Briefly, RNA, 1 μL of random primer and 1 μL of dNTP mixture were brought to 12 μL with RNase-free sterile distilled water and incubated for 5 minutes at 65 °C. Then the mixture Place on ice for at least 1 minute. 4 μL of 5× reaction buffer, 2 μL of 0.1 M DTT and 1 μL of RNaseOUT were then added and the mixture was incubated in a MJ Research thermocycler for 2 minutes at 25 °C. The thermocycler was paused to add 1 μL of Superscript II enzyme and then restarted at 25 ° C for an additional 10 minutes, then switched to 55 ° C for 50 minutes. The reaction was heat inactivated by heating to 70 °C for 15 min and RNA was removed by adding 1 μL of RNase H and incubating at 37 °C for 20 minutes.

簡并PCR反應 Degenerate PCR reaction

對來源於融合瘤細胞之cDNA執行第一輪簡并PCR反應之程序係基於Wang等人(2000)及Co等人(1992)所述之方法。用於此輪之引子(表2)含有有利於選殖至pBluescriptII質體中之限制性位點。 The procedure for performing the first round of degenerate PCR reactions on cDNA derived from fusion tumor cells is based on the method described by Wang et al. (2000) and Co et al. (1992). The primers used in this round (Table 2) contain restriction sites that facilitate selection into the pBluescript II plastid.

將PCR反應組份(表3)在冰上於薄壁PCR管中混合且接著轉移至預加熱及暫停於94℃下之MJ research熱循環儀中。使用來源於Wang等人，2000之程式如下進行反應：名稱：Wang45 The PCR reaction components (Table 3) were mixed on a thin walled PCR tube on ice and then transferred to a MJ research thermocycler preheated and suspended at 94 °C. Use the program from Wang et al., 2000 to react as follows: Name: Wang45

94℃ 3：00min 94°C 3:00min

94℃ 0：15sec 94°C 0:15sec

45℃ 1：00min 45 ° C 1:00min

72℃ 2：00min 72°C 2:00min

轉到2 29次 Go to 2 29 times

72℃ 6：00min 72°C 6:00min

4℃ 總是 4°C always

結束 End

接著使PCR反應混合物在1%低熔點瓊脂糖凝膠上跑膠，將300至400bp帶切除，使用Zymo DNA微型管柱純化，且送至Agencourt biosciences用於定序。使用各別5'及3' PCR引子作為定序引子以自兩個方向產生38SB19可變區cDNA。 The PCR reaction mixture was then run on a 1% low melting agarose gel, the 300 to 400 bp band was excised, purified using a Zymo DNA mini-column and sent to Agencourt biosciences for sequencing. The respective 5' and 3' PCR primers were used as sequencing primers to generate 38SB19 variable region cDNA from both directions.

選殖5'端序列 Colonization of the 5' end sequence

由於用於選殖38SB19可變區輕鏈及重鏈cDNA序列之簡并引子改變5'端序列，因此需要額外定序工作來解密完整序列。使用來自上述方法之初步cDNA序列對38SB19序列所來源之鼠科生殖系序列搜索NCBI IgBlast位點(http：//www.ncbi.nlm.nih.gov/igblast/)。PCR引子經設計(表3)以與鼠科抗體之前導序列黏接以致新PCR反應可產生未由PCR引子改變之完整可變區cDNA。PCR反應、帶純化及定序係如上所述進行。 Since the degenerate primer used to select the 38SB19 variable region light and heavy chain cDNA sequences changes the 5' end sequence, additional sequencing work is required to decrypt the entire sequence. The NCBI IgBlast site (http://www.ncbi.nlm.nih.gov/igblast/) was searched for the murine germline sequence from which the 38SB19 sequence was derived using the preliminary cDNA sequence from the above method. The PCR primers were designed (Table 3) to bind to the murine antibody leader sequence such that the new PCR reaction produced intact variable region cDNA that was not altered by PCR primers. The PCR reaction, band purification and sequencing were carried out as described above.

序列確認之肽分析Sequence confirmation peptide analysis

將可變區之cDNA序列資訊與生殖系恆定區序列組合以獲得全長抗體cDNA序列。接著計算重鏈及輕鏈之分子量且與藉由鼠科38SB19抗體之LC/MS分析所獲得之分子量比較。 The cDNA sequence information of the variable region is combined with the germline constant region sequence to obtain a full length antibody cDNA sequence. The molecular weights of the heavy and light chains were then calculated and compared to the molecular weight obtained by LC/MS analysis of the murine 38SB19 antibody.

表4給出來自38SB19 LC及HC之cDNA序列之計算質量以及藉由LC/MS所量測之值。分子量量測值與38SB19輕鏈及重鏈之cDNA序列相符。 Table 4 gives the calculated masses of the cDNA sequences from 38SB19 LC and HC and the values measured by LC/MS. The molecular weight measurements were consistent with the cDNA sequences of the 38SB19 light and heavy chains.

實例5 Example 5 hu38SB19抗體之重組表現Recombination performance of hu38SB19 antibody

將hu38SB19之可變區序列密碼子優化且藉由Blue Heron Biotechnology來合成。使該等序列側接限制性內切酶位點以便與各別恆定區序列同框選殖於單鏈與串聯雙鏈哺乳動物表現質體中。輕鏈可變區係選殖至ps38SB19LCZv1.0與ps38SB19v1.00質體中之EcoRI及BsiWI位點(圖5A及圖5C)。重鏈可變區係選殖至ps38SB19HCNv1.0與ps38SB19v1.00質體中之HindIII及Apa1位點(圖5B及圖5C)。此等質體可用於以瞬間或穩定轉染在哺乳動物細胞中表現hu38SB19。使用類似表現載體構築體來產生其他嵌合及人類化抗體。 The variable region sequence of hu38SB19 was codon optimized and synthesized by Blue Heron Biotechnology. The sequences are flanked by restriction endonuclease sites for in-frame selection with the respective constant region sequences in single-stranded and tandem double-stranded mammalian expression plastids. The light chain variable region was cloned into the EcoRI and BsiWI sites in the ps38SB19LCZv1.0 and ps38SB19v1.00 plasmids (Fig. 5A and Fig. 5C). The heavy chain variable region was cloned into the HindIII and Apa1 sites in the ps38SB19HCNv1.0 and ps38SB19v1.00 plastids (Fig. 5B and Fig. 5C). These plastids can be used to express hu38SB19 in mammalian cells by transient or stable transfection. Similar expression vector constructs are used to generate additional chimeric and humanized antibodies.

使用來自BD biosciences之CaPO4試劑進行瞬間轉染以在HEK- 293T細胞中表現hu38SB19。對所提供之方案略作修改以增強表現量。簡言之，在轉染前24h將2×106個HEK-293T細胞塗於經聚乙烯亞胺(PEI)塗覆之10cm組織培養盤上。藉由用PBS洗滌細胞且用10mL含1%超低IgG FBS(Hyclone)之DMEM(Invitrogen)替換培養基開始轉染。 在旋轉下將溶液A(10μg DNA，86.8μL Ca2+溶液，且以H2O補至500μL)逐滴添加至溶液B中。將混合物在室溫下培育1min且將1mL混合物逐滴添加至各10cm盤中。轉染後約16h，將培養基用10mL含1%超低IgG FBS之新鮮DMEM替換。約24小時後，將2mM丁酸鈉添加至各10cm盤中。4天後採集轉染。 Transient transfection was performed using CaPO 4 reagent from BD biosciences to express hu38SB19 in HEK-293T cells. Minor changes were made to the proposed solution to enhance performance. Briefly, the 24h prior to transfection 2 × 10 6 HEK-293T cells applied to th (PEI) coated by polyethyleneimine 10cm tissue culture of the disc. Transfection was initiated by washing the cells with PBS and replacing the medium with 10 mL of DMEM (Invitrogen) containing 1% ultra low IgG FBS (Hyclone). Solution A (10 μg of DNA, 86.8 μL of Ca 2+ solution and supplemented with H 2 O to 500 μL) was added dropwise to Solution B under rotation. The mixture was incubated for 1 min at room temperature and 1 mL of the mixture was added dropwise to each 10 cm dish. Approximately 16 h after transfection, the medium was replaced with 10 mL of fresh DMEM containing 1% ultra low IgG FBS. After about 24 hours, 2 mM sodium butyrate was added to each 10 cm dish. Transfection was collected 4 days later.

藉由添加1/10體積之1M Tris/HCl緩衝液(pH 8.0)製備上清液之蛋白質A親和性層析。將pH值經調節之上清液經0.22μm濾膜過濾且裝載於經結合緩衝液(PBS，pH 7.3)平衡之蛋白質A瓊脂糖管柱(HiTrap Protein A HP，1mL，Amersham Biosciences)上。在樣本裝載期間將Q-瓊脂糖前管柱(10mL)連接至蛋白質A管柱之上游以減少細胞物質(諸如DNA)之污染。樣本裝載後，移除前管柱且翻轉蛋白質A管柱之定向進行洗滌及溶離。將管柱用結合緩衝液洗滌直至獲得在280nm下無吸光度之穩定基線。使用0.5mL/min之流動速率，將抗體用含有0.15M NaCl之0.1M乙酸緩衝液(pH 2.8)溶離。收集約0.25mL之溶離份且藉由添加1/10體積之1M Tris/HCl(pH 8.0)將其中和。將峰溶離份相對於PBS透析隔夜兩次且根據OD280下之吸光度量化經純化抗體。亦可使用蛋白質G管柱以略微不同之程序來純化人類化及嵌合抗體。 The protein A affinity chromatography of the supernatant was prepared by adding 1/10 volume of 1 M Tris/HCl buffer (pH 8.0). The pH-adjusted supernatant was filtered through a 0.22 μm filter and loaded on a Protein A Sepharose column (HiTrap Protein A HP, 1 mL, Amersham Biosciences) equilibrated in binding buffer (PBS, pH 7.3). A Q-Sepharose front column (10 mL) was attached upstream of the Protein A column during sample loading to reduce contamination of cellular material such as DNA. After loading the sample, the front column was removed and the orientation of the Protein A column was inverted for washing and dissolving. The column was washed with binding buffer until a stable baseline with no absorbance at 280 nm was obtained. The antibody was eluted with 0.1 M acetate buffer (pH 2.8) containing 0.15 M NaCl using a flow rate of 0.5 mL/min. Approximately 0.25 mL of the fraction was collected and neutralized by the addition of 1/10 volume of 1 M Tris/HCl (pH 8.0). The peak fractions were dialyzed overnight with respect twice with PBS and the absorbance at OD 280 quantization purified antibody. Humanized and chimeric antibodies can also be purified using a protein G column in slightly different procedures.

所有所述嵌合及人類化抗-CD38抗體均係以如上所述之類似程序經表現及純化。 All of the chimeric and humanized anti-CD38 antibodies were expressed and purified in a similar procedure as described above.

實例6Example 6 嵌合抗-CD38抗體之抗體依賴性細胞介導之細胞毒性(ADCC)活性Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of chimeric anti-CD38 antibodies

由於先前已顯示一些抗-CD38抗體作為具有人類IgG1恆定區之嵌 合或人類化抗體之形式具有ADCC及/或CDC活性(J.H.Ellis等人1995,J Immunol,155：925-937；F.K.Stevenson等人1991,Blood,77：1071-1079；WO 2005/103083)，因此製造由鼠科可變區及人類IgG1/Igκ恆定區組成之38SB13、38SB18、38SB19、38SB30、38SB31及38SB39之嵌合型式且測試ADCC及/或CDC活性。 Since some anti-CD38 antibodies have previously been shown to have ADCC and/or CDC activity as chimeric or humanized antibodies with human IgGl constant regions (JHEllis et al. 1995, J Immunol , 155: 925-937; FK Stevenson et al. 1991). , Blood , 77: 1071-1079; WO 2005/103083), thus making a chimeric version of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 consisting of a murine variable region and a human IgG1/Ig kappa constant region and testing ADCC And / or CDC activity.

首先使用Ramos細胞作為靶細胞及使用人類自然殺傷(NK)細胞作為效應細胞測試ch38SB13、ch38SB18、ch38SB19、ch38SB30、ch38SB31及ch38SB39之ADCC。使用乳酸去氫酶(LDH)釋放檢定量測細胞溶解(R.L.Shields等人，2001,J Biol Chem,276：6591-6604)。 ADCCs of ch38SB13, ch38SB18, ch38SB19, ch38SB30, ch38SB31, and ch38SB39 were first tested using Ramos cells as target cells and human natural killer (NK) cells as effector cells. Cell lysis was quantified using lactate dehydrogenase (LDH) release assay (RL Shields et al, 2001, J Biol Chem , 276: 6591-6604).

使用NK分離套組II(Miltenyi Biotech)之修改方案，首先自人類血液(來自正常供體；購自Research Blood Components,Inc.,Brighton,MA)分離NK細胞。將血液用漢克氏(Hank's)平衡鹽溶液(HBSS)稀釋2-3倍。將25mL經稀釋血液小心疊層於50mL錐形管中之25mL Ficoll Paque之上且在19℃下以400g離心45min。自界面收集周邊血液單核細胞(PBMC)，將其轉移至新的50mL錐形管中，且用HBSS洗滌一次。將PBMC再懸浮於2mL NK分離緩衝液中，且接著將500μL生物素-抗體混合液(來自NK分離套組，130-091-152，Miltenyi Biotech)添加至細胞懸浮液中。生物素-抗體混合液含有與淋巴細胞(除NK細胞以外)結合之經結合生物素之抗體。將混合物在4℃下培育10min，且接著添加1.5mL NK分離緩衝液(PBS、0.1% BSA、1mM EDTA)及1mL抗生物素微珠。將細胞-抗體混合物在4℃下再培育15min。接著，將細胞用50mL NK分離緩衝液洗滌一次且再懸浮於3mL NK分離緩衝液中。接著，將MACS LS管柱(在MACS分離器上，Miltenyi Biotech)用3mL NK分離緩衝液預洗滌。接著將細胞懸浮液施加於LS管柱上。將排出液(具有未經標記細胞之溶離份)收集於新的50mL錐形管中。將管柱用3mL NK分離緩衝液洗滌3次。將全部排出液收集於同一管中 且用50mL NK分離緩衝液洗滌一次。將NK細胞塗於30mL補充5%胎牛血清、50μg/mL慶大黴素之RPMI-1640中。 NK cells were first isolated from human blood (from normal donors; purchased from Research Blood Components, Inc., Brighton, MA) using a modification of NK Separation Kit II (Miltenyi Biotech). The blood was diluted 2-3 times with Hank's Balanced Salt Solution (HBSS). 25 mL of diluted blood was carefully stacked on top of 25 mL Ficoll Paque in a 50 mL conical tube and centrifuged at 400 g for 45 min at 19 °C. Peripheral blood mononuclear cells (PBMC) were collected from the interface, transferred to a new 50 mL conical tube, and washed once with HBSS. PBMC were resuspended in 2 mL of NK isolation buffer, and then 500 μL of biotin-antibody mixture (from NK separation kit, 130-091-152, Miltenyi Biotech) was added to the cell suspension. The biotin-antibody mixture contains antibodies bound to biotin in combination with lymphocytes (other than NK cells). The mixture was incubated at 4 °C for 10 min, and then 1.5 mL of NK separation buffer (PBS, 0.1% BSA, 1 mM EDTA) and 1 mL of avidin microbeads were added. The cell-antibody mixture was incubated for an additional 15 min at 4 °C. Next, the cells were washed once with 50 mL of NK separation buffer and resuspended in 3 mL of NK separation buffer. Next, the MACS LS column (on a MACS separator, Miltenyi Biotech) was pre-washed with 3 mL of NK separation buffer. The cell suspension is then applied to the LS column. The effluent (with fractions of unlabeled cells) was collected in a new 50 mL conical tube. The column was washed 3 times with 3 mL of NK separation buffer. Collect all the effluent in the same tube Wash once with 50 mL NK separation buffer. NK cells were applied to 30 mL of RPMI-1640 supplemented with 5% fetal bovine serum and 50 μg/mL gentamicin.

將於補充0.1% BSA、20mM HEPES(pH 7.4)及50μg/mL慶大黴素之RPMI-1640培養基(下文表示為RHBP培養基)中之各種濃度之ch38SB13、ch38SB18、ch38SB19、ch38SB30、ch38SB31及ch38SB39抗體等分(50μL/孔)至圓底96孔盤中。將靶Ramos細胞以106個細胞/毫升再懸浮於RHBP培養基中且添加至含有抗體稀釋液之各孔中(100μL/孔)。將含有靶細胞及抗體稀釋液之盤在37℃下培育30min。接著將NK細胞(50μL/孔)添加至含有靶細胞之孔中，通常添加比率為1個靶細胞/3-6個NK細胞。將RHBP培養基(50μL/孔)添加至含有NK細胞之對照孔中。另外，將20μL Triton X-100溶液(RPMI-1640培養基，10% Triton X-100)添加至僅含有靶細胞而無抗體之3個孔中以測定最大可能LDH釋放。將混合物在37℃下培育4h，接著以1200rpm離心10min，且將100μL上清液小心轉移至新的平底96孔盤中。將細胞毒性偵側套組(Roche 1 644 793)之LDH反應混合物(100μL/孔)添加至各孔中且在室溫下培育5-30min。在490nm下量測樣本之光學密度(OD490)。藉由將100%溶解歸於經Triton X-100處理之樣本的OD490值且將0%溶解歸於僅含有靶細胞之未經處理對照樣本之OD490值來測定各樣本之特異性溶解百分數。僅含有NK細胞之樣本得到可忽略之OD490讀數。 Various concentrations of ch38SB13, ch38SB18, ch38SB19, ch38SB30, ch38SB31 and ch38SB39 antibodies in RPMI-1640 medium supplemented with 0.1% BSA, 20 mM HEPES (pH 7.4) and 50 μg/mL gentamicin (hereinafter referred to as RHBP medium) Aliquot (50 μL/well) into a round bottom 96-well plate. The target Ramos cells were resuspended in RHBP medium at 10 6 cells/ml and added to each well containing the antibody dilution (100 μL/well). The plate containing the target cells and antibody dilutions was incubated at 37 ° C for 30 min. Then, NK cells (50 μL/well) were added to the wells containing the target cells, usually in a ratio of 1 target cell/3-6 NK cells. RHBP medium (50 μL/well) was added to control wells containing NK cells. In addition, 20 μL of Triton X-100 solution (RPMI-1640 medium, 10% Triton X-100) was added to 3 wells containing only target cells without antibodies to determine the maximum possible LDH release. The mixture was incubated at 37 °C for 4 h, then centrifuged at 1200 rpm for 10 min, and 100 μL of the supernatant was carefully transferred to a new flat-bottom 96-well plate. The LDH reaction mixture (100 μL/well) of the cytotoxicity side set (Roche 1 644 793) was added to each well and incubated for 5-30 min at room temperature. The optical density (OD 490 ) of the sample was measured at 490 nm. The percent specific lysis of each sample was determined by assigning 100% dissolution to the OD 490 value of the Triton X-100 treated sample and 0% dissolution to the OD 490 value of the untreated control sample containing only the target cells. Samples containing only NK cells gave negligible OD 490 readings.

當使用Ramos靶細胞及NK效應細胞測試時，嵌合抗-CD38抗體顯示極有效之ADCC活性(圖6)。藉由非線性回歸方法使用S形劑量反應曲線評估EC50值且得到如下值：對於ch38SB13為0.0013μg/mL，對於ch38SB18為0.0013μg/mL，對於ch38SB19為0.0018μg/mL，對於ch38SB30為0.0022μg/mL，對於ch38SB31為0.0012μg/mL，對於ch38SB39為0.1132μg/mL。嵌合抗-CD38抗體亦對LP-1(DSMZ ACC 41)多發性骨髓瘤細胞顯示出有效之ADCC活性(EC50值：對於ch38SB18為0.00056μg/mL；對於ch38SB19為0.00034μg/mL；對於ch38SB31為0.00024μg/mL)(圖7A)。Ch38SB19亦經由ADCC有效殺死Daudi淋巴瘤細胞(圖7B)、NALM-6 B-ALL細胞(DSMZ ACC 128)(圖8A)及MOLT-4 T-ALL細胞(ATTC CRL-1582)(圖8B)，從而表明具有顯著有效細胞凋亡活性之抗-CD38抗體亦具有對抗來源於各種造血惡性疾病之各種腫瘤細胞之有效ADCC活性。另外，非結合性IgG1對照抗體(利妥昔單抗(rituximab)，BiogenIdec)(圖7A、圖8A及圖8B)或mu38SB19(圖7B)在相同實驗中不具有顯著ADCC活性。 The chimeric anti-CD38 antibody showed extremely potent ADCC activity when tested using Ramos target cells and NK effector cells (Fig. 6). By using non-linear regression method of S-shaped dose response curve EC 50 value and the evaluation value obtained as follows: for ch38SB13 of 0.0013μg / mL, for ch38SB18 of 0.0013μg / mL, for ch38SB19 of 0.0018μg / mL, for ch38SB30 of 0.0022μg /mL was 0.0012 μg/mL for ch38SB31 and 0.1132 μg/mL for ch38SB39. The chimeric anti-CD38 antibody also showed potent ADCC activity on LP-1 (DSMZ ACC 41) multiple myeloma cells (EC 50 values: 0.00056 μg/mL for ch38SB18; 0.00034 μg/mL for ch38SB19; for ch38SB31 for ch38SB31) It was 0.00024 μg/mL) (Fig. 7A). Ch38SB19 also effectively killed Daudi lymphoma cells (Fig. 7B), NALM-6 B-ALL cells (DSMZ ACC 128) (Fig. 8A) and MOLT-4 T-ALL cells (ATTC CRL-1582) via ADCC (Fig. 8B). Thus, the anti-CD38 antibody having significant potent apoptotic activity also has potent ADCC activity against various tumor cells derived from various hematopoietic malignant diseases. In addition, the non-binding IgG1 control antibody (rituximab, Biogen Idec) (Fig. 7A, Fig. 8A and Fig. 8B) or mu38SB19 (Fig. 7B) did not have significant ADCC activity in the same experiment.

實例7 Example 7 嵌合抗-CD38抗體之CDC活性 CDC activity of chimeric anti-CD38 antibody

Ch38SB13、ch38SB18、ch38SB19、ch38SB30、ch38SB31及ch38SB39之CDC活性係基於自(H.Gazzano-Santoro等人1997,J.Immunol Methods,202：163-171)修改之方法來量測。人類補體為凍乾人類補體血清(Sigma-Aldrich S1764)，在即將實驗前將其如製造商所指示用無菌純化水復水且接著用RHBP培養基稀釋5倍。將以106個細胞/毫升懸浮於RHBP培養基中之靶細胞等分至平底96孔組織培養盤之孔中(50μL/孔)。接著，添加50μL於RHBP培養基中之各種濃度(10nM至0.001nM)之抗-CD38抗體(每個樣本一種抗體)，隨後添加50μL/孔之補體溶液。接著在37℃下在含有5% CO2之潮濕氣氛中將盤培育2h，其後將50μL於RHBP中稀釋(最終10%)之40% Alamar藍試劑(Biosource DAL1100)添加至各孔中以量測細胞生存力。Alamar藍監測減少存活細胞之能力。將盤在37℃下培育5-18h，隨後在540/590nm下量測螢光(單位為相對螢光單位，RFU)。藉由首先自各樣本之RFU值減去背景RFU值(僅含有培養基而無任何細胞之孔)而相對於背景螢光校正實驗值，且接著以該經校正RFU值除以未經處理之細胞樣本之 經校正RFU值來測定各樣本之特異性細胞生存力之百分數。 The CDC activities of Ch38SB13, ch38SB18, ch38SB19, ch38SB30, ch38SB31 and ch38SB39 are based on methods modified from (H. Gazzano-Santoro et al. 1997, J. Immunol Methods , 202: 163-171). Human complement was lyophilized human complement serum (Sigma-Aldrich S1764), which was reconstituted with sterile purified water as indicated by the manufacturer and then diluted 5 fold with RHBP medium just prior to the experiment. Target cells suspended in RHBP medium at 10 6 cells/ml were aliquoted into wells of a flat-bottom 96-well tissue culture dish (50 μL/well). Next, 50 μL of various concentrations (10 nM to 0.001 nM) of anti-CD38 antibody (one antibody per sample) in RHBP medium was added, followed by addition of 50 μL/well of the complement solution. The plate was then incubated for 2 h at 37 ° C in a humidified atmosphere containing 5% CO 2 , after which 50 μL of 40% Alamar Blue Reagent (Biosource DAL1100) diluted in RHBP (final 10%) was added to each well. Cell viability was measured. Alamar Blue monitors the ability to reduce viable cells. The plates were incubated at 37 ° C for 5-18 h, followed by fluorescence at 540/590 nm (in units of relative fluorescence, RFU). The experimental value is corrected relative to background fluorescence by first subtracting the background RFU value (containing only the medium without any cell pores) from the RFU value of each sample, and then dividing the corrected RFU value by the untreated cell sample The corrected RFU values were used to determine the percentage of specific cell viability of each sample.

當使用最終稀釋度為5%之人類補體以Raji-IMG細胞來測試嵌合抗-CD38抗體樣本之CDC活性時，嵌合抗-CD38抗體顯示極有效之CDC活性(圖9)。Raji-IMG為來源於Raji細胞(ATCC CCL-86)之細胞且表現較低含量之膜補體抑制劑CD55及CD59。藉由非線性回歸自圖8所示之S形劑量反應曲線來評估EC50值且結果如下：對於ch38SB13為0.005μg/mL，對於ch38SB18為0.0101μg/mL，對於ch38SB19為0.028μg/mL，對於ch38SB30為0.020μg/mL，對於ch38SB31為0.010μg/mL，且對於ch38SB39為0.400μg/mL。嵌合抗-CD38抗體亦對LP-1多發性骨髓瘤細胞顯示出有效之CDC活性(EC50值：對於ch38SB18為0.032μg/mL；對於ch38SB19為0.030μg/mL；對於ch38SB31為0.043μg/mL)，而非結合性嵌合對照IgG1(利妥昔單抗，BiogenIdec)不具有任何CDC活性(圖10)。當針對Daudi淋巴瘤細胞測試嵌合CD38抗體時，不同抗-CD38抗體在其CDC活性方面不同(圖11)。在補體存在下與1.25μg/mL ch38SB19一起培育後Daudi細胞之特異性生存力小於15%，而在補體存在下與ch38SB18或ch38SB39(1.25μg/mL或更高濃度)一起培育後此等細胞之特異性生存力僅少量降低(特異性生存力分別為85%及91%)。另外，當在補體存在下將Daudi細胞與1.25μg/mL或更高濃度之ch38SB13、ch38SB30及ch38SB31一起培育時，觀測到特異性生存力僅適度降低(特異性生存力分別為65%、45%及53%)。 When the CDC activity of the chimeric anti-CD38 antibody sample was tested using Raji-IMG cells with human complement at a final dilution of 5%, the chimeric anti-CD38 antibody showed extremely potent CDC activity (Fig. 9). Raji-IMG is a cell derived from Raji cells (ATCC CCL-86) and exhibits lower levels of membrane complement inhibitors, CD55 and CD59. By non-linear regression from the S-shape shown in FIG. 8 to evaluate the dose-response curves and the EC 50 values were as follows: for ch38SB13 was 0.005μg / mL, for ch38SB18 of 0.0101μg / mL, for ch38SB19 of 0.028μg / mL, for Ch38SB30 was 0.020 μg/mL, 0.010 μg/mL for ch38SB31, and 0.400 μg/mL for ch38SB39. Chimeric anti -CD38 antibody also LP-1 multiple myeloma cells showed valid CDC activity (EC 50 values: for ch38SB18 was 0.032μg / mL; for ch38SB19 was 0.030μg / mL; for ch38SB31 was 0.043μg / mL The non-binding chimeric control IgG1 (rituximab, Biogen Idec) did not have any CDC activity (Figure 10). When the chimeric CD38 antibody was tested against Daudi lymphoma cells, the different anti-CD38 antibodies differed in their CDC activity (Figure 11). The specific viability of Daudi cells was less than 15% after incubation with 1.25 μg/mL ch38SB19 in the presence of complement, and after incubation with ch38SB18 or ch38SB39 (1.25 μg/mL or higher) in the presence of complement. Specific viability was only slightly reduced (specific viability was 85% and 91%, respectively). In addition, when Daudi cells were incubated with 238 μg/mL or higher of ch38SB13, ch38SB30 and ch38SB31 in the presence of complement, only a modest decrease in specific viability was observed (specific viability was 65%, 45%, respectively). And 53%).

實例8Example 8 人類化抗-CD38抗體之結合親和力及細胞凋亡、ADCC及CDC活性Binding Affinity and Apoptosis, ADCC and CDC Activity of Humanized Anti-CD38 Antibody

人類化38SB19之兩種型式(hu38SB19 v1.00及v1.20)及嵌合38SB19在使用Ramos細胞測試時顯示相似之結合親和力，KD值分別為0.23nM、0.25nM及0.18nM之(圖12A)。亦在競爭結合檢定中比較嵌合及人類化38SB19抗體之結合親和力，其中量測其與經結合生物素 之鼠科38SB19抗體競爭結合之能力。將經生物素標記之鼠科38SB19抗體(3×10-10M)與各種濃度之ch38SB19、hu38SB19 v1.00或hu38SB19 v1.20混合。將抗體混合物與Ramos細胞一起培育，且藉由FACS分析使用結合FITC之抗生蛋白鏈菌素量測與細胞結合之經結合生物素之鼠科38SB19的量。Hu38SB19 v1.00、hu38SB19 v1.20及ch38SB19充分等同地與經結合生物素之鼠科38SB19競爭結合(圖12B)，從而再次表明結合親和力不受人類化影響。當比較ch38SB19、hu38SB19 v1.00及hu38SB19 v1.20(10-8至10-11M)之誘發Daudi細胞凋亡之能力時，其顯示相似之細胞凋亡活性(圖13)。此外，hu38SB19 v1.00及v1.20在LP-1細胞中亦顯示與ch38SB19相似之ADCC(圖14)且在Raji-IMG及LP-1細胞中顯示與ch38SB19相似之CDC效能(圖15)。Hu38SB19 v1.00在T細胞急性淋巴母細胞白血病細胞株DND-41(DSMZ 525)中亦顯示與ch38SB19相似之CDC活性(圖15)。對hu38SB19 v1.00進一步測試其在一組不同細胞株中誘發細胞凋亡之能力(圖16)。用hu38SB19 v1.00(10-8M)處理使得以下細胞株中之Annexin V陽性細胞顯著增加：B細胞淋巴瘤細胞株SU-DHL-8(DSMZ ACC 573)(自未經處理對照組中之7%至經hu38SB19處理之細胞中之97%)及NU-DUL-1(DSMZ ACC 579)(自未經處理對照組中之10%至經hu38SB19處理之細胞中之37%)及T-ALL細胞株DND-41(自未經處理對照組中之7%至經hu38SB19處理之細胞中之69%)。另外，用hu38SB19 v1.00(10-8M)處理使以下細胞株中之Annexin V陽性細胞之部分增加：B細胞淋巴細胞性白血病細胞株JVM-13(DSMZ ACC 19)(自未經處理對照組中之8%至經hu38SB19處理之細胞中之17%)及毛細胞白血病細胞株HC-1(DSMZ ACC 301)(自未經處理對照組中之6%至經hu38SB19處理之細胞中之10%)。 The two types of humanized 38SB19 (hu38SB19 v1.00 and v1.20) and chimeric 38SB19 showed similar binding affinities when tested using Ramos cells with K D values of 0.23 nM, 0.25 nM and 0.18 nM, respectively (Figure 12A). ). The binding affinity of the chimeric and humanized 38SB19 antibodies was also compared in a competition binding assay in which the ability to compete for binding to the murine 38SB19 antibody bound to biotin was measured. Biotinylated murine 38SB19 antibody (3 x 10 -10 M) was mixed with various concentrations of ch38SB19, hu38SB19 v1.00 or hu38SB19 v1.20. The antibody mixture was incubated with Ramos cells and the amount of murine-bound biotinylated murine 38SB19 bound to cells was measured by FACS analysis using FITC-conjugated streptavidin. Hu38SB19 v1.00, hu38SB19 v1.20 and ch38SB19 competed fully with the binding biotinylated murine 38SB19 (Fig. 12B), again indicating that the binding affinity was not affected by humanization. When comparing the ability of ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 (10 -8 to 10 -11 M) to induce apoptosis of Daudi cells, it showed similar apoptotic activity (Fig. 13). In addition, hu38SB19 v1.00 and v1.20 also showed ADCC similar to ch38SB19 in LP-1 cells (Fig. 14) and showed similar CDC potency as ch38SB19 in Raji-IMG and LP-1 cells (Fig. 15). Hu38SB19 v1.00 also showed similar CDC activity to ch38SB19 in T cell acute lymphoblastic leukemia cell line DND-41 (DSMZ 525) (Fig. 15). Hu38SB19 v1.00 was further tested for its ability to induce apoptosis in a panel of different cell lines (Figure 16). Treatment with hu38SB19 v1.00 (10 -8 M) resulted in a significant increase in Annexin V positive cells in the following cell lines: B cell lymphoma cell line SU-DHL-8 (DSMZ ACC 573) (from untreated control group) 7% to 97% of hu38SB19-treated cells and NU-DUL-1 (DSMZ ACC 579) (from 10% in untreated controls to 37% of cells treated with hu38SB19) and T-ALL Cell line DND-41 (from 7% in the untreated control to 69% in the cells treated with hu38SB19). In addition, treatment with hu38SB19 v1.00 (10 -8 M) increased the portion of Annexin V-positive cells in the following cell lines: B-cell lymphocytic leukemia cell line JVM-13 (DSMZ ACC 19) (from untreated control) 8% of the cells in the group (17% of the cells treated with hu38SB19) and the hairy cell leukemia cell line HC-1 (DSMZ ACC 301) (from 6% in the untreated control group to 10 in the hu38SB19-treated cells) %).

類似地，人類化38SB31之兩種型式(hu38SB31 v1.1及v1.2)及嵌合38SB31在使用Ramos細胞測試時顯示相似之結合親和力，KD值分別為 0.13nM、0.11nM及0.12nM。如上文所述亦在競爭結合檢定中比較嵌合及人類化38SB31抗體之結合親和力且其表現充分等同。對hu38SB31 v1.1進一步測試其在數種細胞株中誘發細胞凋亡之能力。 人類化抗體對Ramos、Daudi、Molp-8及SU-DHL-8細胞顯示與ch38SB31相似之細胞凋亡活性。此外，hu38SB31 v1.1在此等細胞株中亦顯示與ch38SB31相似之ADCC及CDC活性。 Similarly, two types of humanized 38SB31 (hu38SB31 v1.1 and V1.2) and the fitting 38SB31 using Ramos cells when the test showed similar binding affinities, K D values of 0.13nM, 0.11nM, and 0.12nM. The binding affinities of the chimeric and humanized 38SB31 antibodies were also compared in the competition binding assay as described above and their performance was sufficiently equivalent. Hu38SB31 v1.1 was further tested for its ability to induce apoptosis in several cell lines. The humanized antibody showed apoptotic activity similar to that of ch38SB31 to Ramos, Daudi, Molp-8, and SU-DHL-8 cells. In addition, hu38SB31 v1.1 also showed similar ADCC and CDC activities to ch38SB31 in these cell lines.

實例9 Example 9 38SB13、38SB18、38SB19、38SB30、38SB31及38SB39之活體內功效In vivo efficacy of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39

在使用Ramos淋巴瘤細胞確立之免疫缺乏小鼠(雌性CB.17 SCID)中之存活人類異種移植腫瘤模型中研究38SB13、38SB18、38SB19、38SB30、38SB31及38SB39之活體內抗腫瘤活性。將雌性CB.17 SCID小鼠用於0.1mL無血清培養基中之2×106個Ramos細胞經側尾靜脈接種。腫瘤細胞接種後7天，將小鼠基於體重隨機分成7組。除具有6隻小鼠之經38SB31處理之組及具有8隻小鼠之經38SB39處理之組以外，每組有10隻小鼠。以40mg/kg之劑量，每週兩次，在三個連續週內，在細胞接種後7天開始，將抗體經靜脈內給予小鼠。若一或兩隻後肢麻痺，體重自預處理值降低20%以上或動物過度病態而不能進食及進水，則處死小鼠。與經PBS處理之小鼠相比，用38SB13、38SB18、38SB19、38SB30、38SB31或38SB39處理顯著延長小鼠存活(圖17)。經PBS處理之小鼠之中值存活期為22天且經抗體處理之組之中值存活期在28至33天之範圍內。 The in vivo antitumor activity of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 was investigated in a surviving human xenograft tumor model in immunodeficient mice (female CB.17 SCID) established using Ramos lymphoma cells. Female CB.17 SCID mice were used in 0.1mL serum-free medium of th 2 × 10 6 Ramos cells inoculated intravenously via the lateral tail. Seven days after tumor cell inoculation, mice were randomly divided into 7 groups based on body weight. Except for the 38SB31 treated group with 6 mice and the 38SB39 treated group with 8 mice, there were 10 mice in each group. The antibody was administered intravenously to the mice at a dose of 40 mg/kg twice a week for three consecutive weeks starting 7 days after cell seeding. If one or two hind limbs are paralyzed, the body weight is reduced by more than 20% from the pretreatment value, or the animal is overly ill and cannot eat and enter the water, the mice are sacrificed. Treatment with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 significantly prolonged mouse survival compared to PBS treated mice (Figure 17). The median survival of PBS-treated mice was 22 days and the median survival of the antibody-treated groups ranged from 28 to 33 days.

在免疫缺乏小鼠中之其他人類異種移植腫瘤模型中進一步研究mu38SB19及hu38SB19之活體內抗腫瘤活性。對於Daudi淋巴瘤存活模型，將SCID小鼠用於0.1mL無血清培養基中之5×106個Daudi細胞經側尾靜脈接種。如上所述進行研究。與經PBS處理之小鼠相比，用 mu38SB19或hu38SB19處理顯著延長小鼠存活(圖18)。經PBS處理之小鼠之中值存活期為22天，而經抗體處理之小鼠之中值存活期為47天。 The in vivo antitumor activity of mu38SB19 and hu38SB19 was further investigated in other human xenograft tumor models in immunodeficient mice. For Daudi lymphoma survival model, SCID mice were used for the serum-free medium 0.1mL of th 5 × 10 6 Daudi cells were inoculated via the lateral tail vein. The study was carried out as described above. Treatment with mu38SB19 or hu38SB19 significantly prolonged mouse survival compared to PBS treated mice (Figure 18). The median survival of PBS-treated mice was 22 days, while the antibody-treated mice had a median survival of 47 days.

對於NCI-H929多發性骨髓瘤腫瘤模型，將SCID小鼠用107個細胞經皮下接種。當腫瘤在第6天可觸知時，將動物根據體重隨機分成10組且開始抗體處理。以40mg/kg之劑量，每週兩次，在三個連續週內，將hu38SB19抗體或非結合性嵌合IgG1對照抗體(利妥昔單抗，BiogenIdec)經靜脈內給予小鼠。監測腫瘤體積，且若腫瘤尺寸達到2000mm3或腫瘤壞死，則處死動物。經PBS處理之組在第89天達到1000mm3之平均腫瘤體積，嵌合IgG1對照抗體組在第84天達到此平均腫瘤體積(圖19)。用hu38SB19處理在所有10隻動物中完全阻止腫瘤生長。相比之下，在經PBS處理之組中僅有兩隻動物且在嵌合IgG1對照抗體組中有三隻動物顯示腫瘤衰退。 For the NCI-H929 multiple myeloma tumor model, SCID mice were inoculated subcutaneously with 10 7 cells. When the tumor was palpable on day 6, the animals were randomly divided into 10 groups according to body weight and antibody treatment was started. The hu38SB19 antibody or the non-binding chimeric IgG1 control antibody (rituximab, Biogen Idec) was administered intravenously to mice at a dose of 40 mg/kg twice a week for three consecutive weeks. Tumor volume was monitored, and if the tumor size reached 2000 mm 3 or tumor necrosis, the animals were sacrificed. The PBS-treated group reached an average tumor volume of 1000 mm 3 on day 89, and the chimeric IgG1 control antibody group reached this mean tumor volume on day 84 (Figure 19). Treatment with hu38SB19 completely prevented tumor growth in all 10 animals. In contrast, only two animals in the PBS-treated group and three animals in the chimeric IgG1 control antibody group showed tumor regression.

對於MOLP-8多發性骨髓瘤腫瘤模型，將SCID小鼠用107個細胞經皮下接種。當腫瘤在第4天可觸知時，將動物根據體重隨機分成10組且開始抗體處理。以40mg/kg之劑量，每週兩次，在三個連續週內，將hu38SB19及mu38SB19抗體或嵌合IgG1對照抗體經靜脈內給予小鼠。監測腫瘤體積，且若腫瘤尺寸達到2000mm3或腫瘤壞死，則處死動物。經PBS處理之組在第22天達到500mm3之平均腫瘤體積，嵌合IgG1對照抗體組在第23天達到此平均腫瘤體積(圖20)。在此等組中無一腫瘤衰退。相比之下，用hu38SB19或mu38SB19處理分別使10隻動物中之8隻或10隻動物中之6隻腫瘤衰退。 For the MOLP-8 multiple myeloma tumor model, SCID mice were inoculated subcutaneously with 10 7 cells. When the tumor was palpable on day 4, the animals were randomly divided into 10 groups according to body weight and antibody treatment was started. The hu38SB19 and mu38SB19 antibodies or the chimeric IgG1 control antibody were administered intravenously to mice at a dose of 40 mg/kg twice a week for three consecutive weeks. Tumor volume was monitored, and if the tumor size reached 2000 mm 3 or tumor necrosis, the animals were sacrificed. The PBS-treated group reached an average tumor volume of 500 mm 3 on day 22, and the chimeric IgG1 control antibody group reached this mean tumor volume on day 23 (Figure 20). None of the tumors in this group declined. In contrast, treatment with hu38SB19 or mu38SB19 caused tumor regression in 8 out of 10 animals or 6 animals, respectively.

表格form

<110> 法商賽諾菲-安萬特公司 <110> French company Sanofi-Aventis

<120> 用於治療癌症之新穎抗-CD38抗體 <120> Novel anti-CD38 antibody for the treatment of cancer

<130> FR2006-043 <130> FR2006-043

<140> 096139053 <140> 096139053

<141> 2007-10-18 <141> 2007-10-18

<150> 06291628-3 <150> 06291628-3

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<213> 小鼠 <213> mouse

<220> <220>

<221> CDS <221> CDS

<222> (1)..(342) <222> (1)..(342)

<400> 51 <400> 51

<210> 52 <210> 52

<211> 114 <211> 114

<212> PRT <212> PRT

<213> 小鼠 <213> mouse

<400> 52 <400> 52

<210> 53 <210> 53

<211> 360 <211> 360

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<220> <220>

<221> CDS <221> CDS

<222> (1)..(360) <222> (1)..(360)

<400> 53 <400> 53

<210> 54 <210> 54

<211> 120 <211> 120

<212> PRT <212> PRT

<213> 小鼠 <213> mouse

<400> 54 <400> 54

<210> 55 <210> 55

<211> 357 <211> 357

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<220> <220>

<221> CDS <221> CDS

<222> (1)..(357) <222> (1)..(357)

<400> 55 <400> 55

<210> 56 <210> 56

<211> 119 <211> 119

<212> PRT <212> PRT

<213> 小鼠 <213> mouse

<400> 56 <400> 56

<210> 57 <210> 57

<211> 351 <211> 351

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<220> <220>

<221> CDS <221> CDS

<222> (1)..(351) <222> (1)..(351)

<400> 57 <400> 57

<210> 58 <210> 58

<211> 117 <211> 117

<212> PRT <212> PRT

<213> 小鼠 <213> mouse

<400> 58 <400> 58

<210> 59 <210> 59

<211> 360 <211> 360

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<220> <220>

<221> CDS <221> CDS

<222> (1)..(360) <222> (1)..(360)

<400> 59 <400> 59

<210> 60 <210> 60

<211> 120 <211> 120

<212> PRT <212> PRT

<213> 小鼠 <213> mouse

<400> 60 <400> 60

<210> 61 <210> 61

<211> 324 <211> 324

<212> DNA <212> DNA

<213> 人類 <213> Human

<220> <220>

<221> CDS <221> CDS

<222> (1)..(324) <222> (1)..(324)

<400> 61 <400> 61

<210> 62 <210> 62

<211> 108 <211> 108

<212> PRT <212> PRT

<213> 人類 <213> Human

<400> 62 <400> 62

<210> 63 <210> 63

<211> 324 <211> 324

<212> DNA <212> DNA

<213> 人類 <213> Human

<220> <220>

<221> CDS <221> CDS

<222> (1)..(324) <222> (1)..(324)

<400> 63 <400> 63

<210> 64 <210> 64

<211> 108 <211> 108

<212> PRT <212> PRT

<213> 人類 <213> Human

<400> 64 <400> 64

<210> 65 <210> 65

<211> 360 <211> 360

<212> DNA <212> DNA

<213> 人類 <213> Human

<220> <220>

<221> CDS <221> CDS

<222> (1)..(360) <222> (1)..(360)

<400> 65 <400> 65

<210> 66 <210> 66

<211> 120 <211> 120

<212> PRT <212> PRT

<213> 人類 <213> Human

<400> 66 <400> 66

<210> 67 <210> 67

<211> 324 <211> 324

<212> DNA <212> DNA

<213> 人類 <213> Human

<220> <220>

<221> CDS <221> CDS

<222> (1)..(324) <222> (1)..(324)

<400> 67 <400> 67

<210> 68 <210> 68

<211> 108 <211> 108

<212> PRT <212> PRT

<213> 人類 <213> Human

<400> 68 <400> 68

<210> 69 <210> 69

<211> 324 <211> 324

<212> DNA <212> DNA

<213> 人類 <213> Human

<220> <220>

<221> CDS <221> CDS

<222> (1)..(324) <222> (1)..(324)

<400> 69 <400> 69

<210> 70 <210> 70

<211> 108 <211> 108

<212> PRT <212> PRT

<213> 人類 <213> Human

<400> 70 <400> 70

<210> 71 <210> 71

<211> 351 <211> 351

<212> DNA <212> DNA

<213> 人類 <213> Human

<220> <220>

<221> CDS <221> CDS

<222> (1)..(351) <222> (1)..(351)

<400> 71 <400> 71

<210> 72 <210> 72

<211> 117 <211> 117

<212> PRT <212> PRT

<213> 人類 <213> Human

<400> 72 <400> 72

<210> 73 <210> 73

<211> 36 <211> 36

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<400> 73 <400> 73

<210> 74 <210> 74

<211> 32 <211> 32

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<400> 74 <400> 74

<210> 75 <210> 75

<211> 32 <211> 32

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<220> <220>

<221> misc_feature <221> misc_feature

<222> (1)..(32) <222> (1)..(32)

<223> 混合鹼基係如下定義：H=A+T+C，S=G+C，Y=C+T，K=G+T，M=A+C，R=A+G，W=A+T，V=A+C+G，N=A+C+G+T <223> Mixed bases are defined as follows: H = A + T + C, S = G + C, Y = C + T, K = G + T, M = A + C, R = A + G, W = A+T, V=A+C+G, N=A+C+G+T

<400> 75 <400> 75

<210> 76 <210> 76

<211> 35 <211> 35

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<220> <220>

<221> misc_feature <221> misc_feature

<222> (1)..(35) <222> (1)..(35)

<223> 混合鹼基係如下定義：H=A+T+C，S=G+C，Y=C+T，K=G+T，M=A+C，R=A+G，W=A+T，V=A+C+G，N=A+C+G+T <223> Mixed bases are defined as follows: H = A + T + C, S = G + C, Y = C + T, K = G + T, M = A + C, R = A + G, W = A+T, V=A+C+G, N=A+C+G+T

<400> 76 <400> 76

<210> 77 <210> 77

<211> 31 <211> 31

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<220> <220>

<221> misc_feature <221> misc_feature

<222> (1)..(31) <222> (1)..(31)

<223> 混合鹼基係如下定義：H=A+T+C，S=G+C，Y=C+T，K=G+T，M=A+C，R=A+G，W=A+T，V=A+C+G，N=A+C+G+T <223> Mixed bases are defined as follows: H = A + T + C, S = G + C, Y = C + T, K = G + T, M = A + C, R = A + G, W = A+T, V=A+C+G, N=A+C+G+T

<400> 77 <400> 77

<210> 78 <210> 78

<211> 46 <211> 46

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<400> 78 <400> 78

<210> 79 <210> 79

<211> 21 <211> 21

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<400> 79 <400> 79

<210> 80 <210> 80

<211> 32 <211> 32

<212> DNA <212> DNA

<213> 小鼠 <213> mouse

<400> 80 <400> 80

<210> 81 <210> 81

<211> 17 <211> 17

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<400> 81 <400> 81

Claims (51)

一種特異性結合CD38之抗體或其抗原決定基(epitope)結合片段，其特徵在於該抗體或其抗原決定基結合片段能夠經由細胞凋亡(apoptosis)、抗體依賴性細胞介導之細胞毒性(ADCC)及補體依賴性細胞毒性(CDC)殺死CD38+細胞，及其中該抗體包含至少一個重鏈及至少一個輕鏈，其中(i)該重鏈包含三個連續互補決定區具有由SEQ ID NO：1、2及3表示之胺基酸序列，且其中該輕鏈包含三個連續互補決定區具有由SEQ ID NO：4、5及6表示之胺基酸序列；(ii)該重鏈包含三個連續互補決定區具有由SEQ ID NO：7、8及9表示之胺基酸序列，且其中該輕鏈包含三個連續互補決定區具有由SEQ ID NO：10、11及12表示之胺基酸序列；(iv)該重鏈包含三個連續互補決定區具有由SEQ ID NO：19、20及21表示之胺基酸序列，且其中該輕鏈包含三個連續互補決定區具有由SEQ ID NO：22、23及24表示之胺基酸序列；(v)該重鏈包含三個連續互補決定區具有由SEQ ID NO：25、26及27表示之胺基酸序列，且其中該輕鏈包含三個連續互補決定區具有由SEQ ID NO：28、29及30表示之胺基酸序列；或(vi)該重鏈包含三個連續互補決定區具有由SEQ ID NO：31、32及33表示之胺基酸序列，且其中該輕鏈包含三個連續互補決定區具有由SEQ ID NO：34、35及36表示之胺基酸序列。 An antibody or an epitope binding fragment thereof that specifically binds to CD38, characterized in that the antibody or its epitope-binding fragment is capable of undergoing apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC) And complement dependent cytotoxicity (CDC) kills CD38 + cells, and wherein the antibody comprises at least one heavy chain and at least one light chain, wherein (i) the heavy chain comprises three consecutive complementarity determining regions having SEQ ID NO The amino acid sequence represented by 1, 2 and 3, and wherein the light chain comprises three consecutive complementarity determining regions having the amino acid sequence represented by SEQ ID NOS: 4, 5 and 6; (ii) the heavy chain comprises The three consecutive complementarity determining regions have an amino acid sequence represented by SEQ ID NOS: 7, 8, and 9, and wherein the light chain comprises three consecutive complementarity determining regions having an amine represented by SEQ ID NOS: 10, 11 and (iv) the heavy chain comprising three consecutive complementarity determining regions having the amino acid sequence represented by SEQ ID NOS: 19, 20 and 21, and wherein the light chain comprises three consecutive complementarity determining regions with SEQ ID NO: amino acid sequence represented by 22, 23 and 24; (v) the weight The chain comprises three consecutive complementarity determining regions having the amino acid sequence represented by SEQ ID NOS: 25, 26 and 27, and wherein the light chain comprises three consecutive complementarity determining regions represented by SEQ ID NOs: 28, 29 and 30 The amino acid sequence; or (vi) the heavy chain comprising three consecutive complementarity determining regions having the amino acid sequence represented by SEQ ID NOS: 31, 32 and 33, and wherein the light chain comprises three consecutive complementarity determining regions There are amino acid sequences represented by SEQ ID NOS: 34, 35 and 36. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段能夠在無基質細胞或基質衍生之細胞激素(cytokines)存在下經由細胞凋亡殺死該CD38+細胞。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof is capable of killing the CD38 via apoptosis in the absence of stromal cells or matrix-derived cytokines + cells. 如請求項1-2中任一項之抗體或其抗原決定基結合片段，其特徵 在於該抗體或其抗原決定基結合片段為單株抗體。 The antibody or epitope-binding fragment thereof according to any one of claims 1 to 2, characterized in that The antibody or its epitope-binding fragment is a monoclonal antibody. 如請求項1-2中任一項之抗體或其抗原決定基結合片段，其特徵在於該CD38+細胞為淋巴瘤細胞、白血病細胞或多發性骨髓瘤細胞。 The antibody or epitope-binding fragment thereof according to any one of claims 1 to 2, wherein the CD38 + cell is a lymphoma cell, a leukemia cell or a multiple myeloma cell. 如請求項4之抗體或其抗原決定基結合片段，其特徵在於該CD38+細胞為非霍奇金氏淋巴瘤(non-Hodgkin's lymphoma，NHL)細胞、伯基特氏淋巴瘤(Burkitt's lymphoma，BL)細胞、多發性骨髓瘤(MM)細胞、慢性B淋巴細胞性白血病(B-CLL)細胞、急性B及T淋巴細胞性白血病(ALL)細胞、T細胞淋巴瘤(TCL)細胞、急性骨髓性白血病(AML)細胞、毛細胞白血病(HCL)細胞、霍奇金氏淋巴瘤(Hodgkin's Lymphoma，HL)細胞或慢性骨髓性白血病(CML)細胞。 The antibody of claim 4 or an epitope binding fragment thereof, characterized in that the CD38 + cell is a non-Hodgkin's lymphoma (NHL) cell or a Burkitt's lymphoma (BL) Cells, multiple myeloma (MM) cells, chronic B lymphocytic leukemia (B-CLL) cells, acute B and T lymphocytic leukemia (ALL) cells, T-cell lymphoma (TCL) cells, acute myeloid Leukemia (AML) cells, hairy cell leukemia (HCL) cells, Hodgkin's Lymphoma (HL) cells, or chronic myeloid leukemia (CML) cells. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段能夠在無基質細胞或基質衍生之細胞激素存在下經由細胞凋亡殺死至少24%之Daudi淋巴瘤細胞。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or its epitope-binding fragment is capable of killing at least 24% of Daudi via apoptosis in the absence of stromal cells or matrix-derived cytokines. Lymphoma cells. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段能夠在無基質細胞或基質衍生之細胞激素存在下經由細胞凋亡殺死7%以上之Ramos淋巴瘤細胞。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or its epitope-binding fragment is capable of killing more than 7% of Ramos via apoptosis in the absence of stromal cells or matrix-derived cytokines Lymphoma cells. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段能夠在無基質細胞或基質衍生之細胞激素存在下經由細胞凋亡殺死11%以上之MOLP-8多發性骨髓瘤細胞。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or its epitope-binding fragment is capable of killing more than 11% of MOLP via apoptosis in the absence of stromal cells or matrix-derived cytokines -8 multiple myeloma cells. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段能夠在無基質細胞或基質衍生之細胞激素存在下經由細胞凋亡殺死36%以上之SU-DHL-8淋巴瘤細胞。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or its epitope-binding fragment is capable of killing more than 36% of SU via apoptosis in the absence of stromal cells or matrix-derived cytokines - DHL-8 lymphoma cells. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段能夠在無基質細胞或基質衍生之細胞激素存在下經由細胞凋亡殺死62%以上之DND-41白血病細胞。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or its epitope-binding fragment is capable of killing more than 62% of DND via apoptosis in the absence of stromal cells or matrix-derived cytokines -41 leukemia cells. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段能夠在無基質細胞或基質衍生之細胞激素存在下經由細胞凋亡殺死27%以上之NU-DUL-1淋巴瘤細胞。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or its epitope-binding fragment is capable of killing 27% or more of NU via apoptosis in the absence of stromal cells or matrix-derived cytokines -DUL-1 lymphoma cells. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段能夠在無基質細胞或基質衍生之細胞激素存在下經由細胞凋亡殺死9%以上之JVM-13白血病細胞。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or its epitope-binding fragment is capable of killing more than 9% of the JVM via apoptosis in the absence of stromal cells or matrix-derived cytokines -13 leukemia cells. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段能夠在無基質細胞或基質衍生之細胞激素存在下經由細胞凋亡殺死4%以上之HC-1白血病細胞。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or its epitope-binding fragment is capable of killing 4% or more of HC via apoptosis in the absence of stromal cells or matrix-derived cytokines -1 leukemia cells. 如請求項1-2及6-13中任一項之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段以3×10-9M或更低之kD結合CD38。 The antibody or epitope-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody or the epitope-binding fragment thereof is bound by k D of 3 × 10 -9 M or lower. CD38. 如請求項1之抗體或其抗原決定基結合片段，其包含如請求項1之(i)所界定特徵，且其特徵在於該抗體或其抗原決定基結合片段包含一個具有由SEQ ID NO：38組成之胺基酸序列之輕鏈可變區，及包含一個具有由SEQ ID NO：50組成之胺基酸序列之重鏈可變區。 The antibody of claim 1, or an epitope binding fragment thereof, comprising the feature as defined in (i) of claim 1 and characterized in that the antibody or antigenic epitope binding fragment thereof comprises one having SEQ ID NO: 38 A light chain variable region of the amino acid sequence consisting of, and a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 50. 如請求項1之抗體或其抗原決定基結合片段，其包含如請求項1之(ii)所界定特徵，且其特徵在於該抗體或其抗原決定基結合片段包含一個具有由SEQ ID NO：40組成之胺基酸序列之輕鏈可變區，及包含一個具有由SEQ ID NO：52組成之胺基酸序列之重鏈可變區。 The antibody of claim 1, or an epitope binding fragment thereof, comprising the feature as defined in claim 1 (ii), and characterized in that the antibody or antigenic epitope binding fragment thereof comprises one having SEQ ID NO: 40 A light chain variable region of the amino acid sequence consisting of, and a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 52. 如請求項1之抗體或其抗原決定基結合片段，其包含如請求項1之(iv)所界定特徵，且其特徵在於該抗體或其抗原決定基結合片段包含一個具有由SEQ ID NO：44組成之胺基酸序列之輕鏈可變區，及包含一個具有由SEQ ID NO：56組成之胺基酸序列之重鏈可變區。 An antibody according to claim 1, wherein the antibody or antigenic determinant binding fragment thereof comprises a SEQ ID The light chain variable region of the amino acid sequence consisting of, and comprising a heavy chain variable region having the amino acid sequence consisting of SEQ ID NO:56. 如請求項1之抗體或其抗原決定基結合片段，其包含如請求項1之(v)所界定特徵，且其特徵在於該抗體或其抗原決定基結合片段包含一個具有由SEQ ID NO：46組成之胺基酸序列之輕鏈可變區，及包含一個具有由SEQ ID NO：58組成之胺基酸序列之重鏈可變區。 The antibody of claim 1, or an epitope binding fragment thereof, comprising the feature as defined in (v) of claim 1 and characterized in that the antibody or antigenic epitope binding fragment thereof comprises one having SEQ ID NO: 46 A light chain variable region of the amino acid sequence consisting of, and a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO:58. 如請求項1之抗體或其抗原決定基結合片段，其包含如請求項1之(vi)所界定特徵，且其特徵在於該抗體或其抗原決定基結合片段包含一個具有由SEQ ID NO：48組成之胺基酸序列之輕鏈可變區，及包含一個具有由SEQ ID NO：60組成之胺基酸序列之重鏈可變區。 The antibody of claim 1, or an epitope binding fragment thereof, comprising the feature as defined in claim 1 (vi), and characterized in that the antibody or antigenic epitope binding fragment thereof comprises one having SEQ ID NO: 48 A light chain variable region of the amino acid sequence consisting of, and a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO:60. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段包含至少一個人類恆定區。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or antigenic epitope binding fragment thereof comprises at least one human constant region. 如請求項20之抗體或其抗原決定基結合片段，其特徵在於該恆定區為人類IgG1/Igκ恆定區。 The antibody of claim 20, or an epitope binding fragment thereof, characterized in that the constant region is a human IgG1/Ig kappa constant region. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體或其抗原決定基結合片段為人類化或表面重塑(resurfaced)抗體。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or its epitope binding fragment is a humanized or surface remodeled antibody. 如請求項22之抗體或其抗原決定基結合片段，其特徵在於該人類化或表面重塑抗體或其抗原決定基結合片段包含一個具有由SEQ ID NO：72表示之胺基酸序列之重鏈可變區，及包含一個具有選自SEQ ID NO：68及70之群之胺基酸序列之輕鏈可變區。 The antibody or antigenic epitope binding fragment thereof of claim 22, wherein the humanized or surface remodeling antibody or antigenic epitope binding fragment thereof comprises a heavy chain having the amino acid sequence represented by SEQ ID NO: 72 A variable region, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 and 70. 如請求項1之抗體或其抗原決定基結合片段，其特徵在於該抗體 或其抗原決定基結合片段為一個Fab、Fab'、F(ab')2或Fv片段。 The antibody of claim 1, or an epitope binding fragment thereof, characterized in that the antibody or its epitope binding fragment is a Fab, Fab', F(ab') 2 or Fv fragment. 一種結合物，其包含如請求項1至24中任一項之抗體或其抗原決定基結合片段與一種細胞毒性劑連接。 A conjugate comprising the antibody of any one of claims 1 to 24, or an epitope binding fragment thereof, linked to a cytotoxic agent. 如請求項25之結合物，其特徵在於該細胞毒性劑係選自由美登素類化合物(maytansinoid)、小分子藥物、茅屋黴素(tomaymycin)衍生物、細黴素(leptomycin)衍生物、前藥、紫杉類藥物(taxoid)、CC-1065及CC-1065類似物組成之群。 A combination according to claim 25, characterized in that the cytotoxic agent is selected from the group consisting of maytansinoids, small molecule drugs, tomaymycin derivatives, leptomycin derivatives, and the like. A group of drugs, taxoids, CC-1065, and CC-1065 analogs. 如請求項26之結合物，其特徵在於該細胞毒性劑為下式之美登素(maytansine)DM1： The combination of claim 26, characterized in that the cytotoxic agent is a maytansine DM1 of the formula: 如請求項26之結合物，其特徵在於該細胞毒性劑為下式之美登素DM4： The combination of claim 26, characterized in that the cytotoxic agent is the genus dinosaur DM4 of the formula: 如請求項26之結合物，其特徵在於該細胞毒性劑為選自由以下 組成之群的茅屋黴素衍生物：8,8'-[1,3-苯二基雙(亞甲氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[5-甲氧基-1,3-苯二基雙(亞甲氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[1,5-戊烷二基雙(氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[1,4-丁烷二基雙(氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[3-甲基-1,5-戊烷二基雙(氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[2,6-吡啶二基雙(氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[4-(3-第三丁氧基羰基胺基丙氧基)-2,6-吡啶二基雙-(亞甲氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[5-(3-胺基丙氧基)-1,3-苯二基雙(亞甲氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[5-(N-甲基-3-第三丁氧基羰基胺基丙基)-1,3-苯二基雙-(亞甲氧基)]-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-{5-[3-(4-甲基-4-甲基二硫基(disulfanyl)-戊醯基胺基)丙氧基]-1,3-苯二基雙(亞甲氧基)}-雙[(S)-2-亞乙-(E)-基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[5-乙醯基硫基甲基-1,3-苯二基雙(亞甲氧基)]-雙[(S)-2-亞 甲基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]雙-{2-[(S)-2-亞甲基-7-甲氧基-5-側氧基(oxo)-1,3,,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-8-基氧基]-乙基}-胺基甲酸第三丁酯8,8'-[3-(2-乙醯基硫基乙基)-1,5-戊烷二基雙(氧基)]-雙[(S)-2-亞甲基-7-甲氧基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[5-(N-4-巰基-4,4-二甲基丁醯基)胺基-1,3-苯二基雙(亞甲氧基)]-雙[7-甲氧基-2-亞甲基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[5-(N-4-甲基二硫基-4,4-二甲基丁醯基)-胺基-1,3-苯二基雙(亞甲氧基)]-雙[7-甲氧基-2-亞甲基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[5-(N-甲基-N-(2-巰基-2,2-二甲基乙基)胺基-1,3-苯二基(亞甲氧基)]-雙[7-甲氧基-2-亞甲基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[5-(N-甲基-N-(2-甲基二硫基-2,2-二甲基乙基)胺基-1,3-苯二基(亞甲氧基)]-雙[7-甲氧基-2-亞甲基-1,2,3,11a-四氫-5H-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(4-(2-(4-巰基-4-甲基)-戊醯胺基-乙氧基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(1-(2-(4-甲基-4-甲基二硫基)-戊醯胺基-乙氧基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(4-(3-(4-甲基-4-甲基二硫基)-戊醯胺基-丙氧基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(4-(4-(4-甲基-4-甲基二硫基)-戊醯胺基-丁氧基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(4-(3-[4-(4-甲基-4-甲基二硫基-戊醯基)-哌嗪-1-基]-丙基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(1-(3-[4-(4-甲基-4-甲基二硫基-戊醯基)-哌嗪-1-基]-丙基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(4-(2-{2-[2-(4-甲基-4-甲基二硫基-戊醯基胺基)-乙氧基]-乙氧基}-乙氧基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(1-(2-{2-[2-(2-{2-[2-(4-甲基-4-甲基二硫基-戊醯基胺基)-乙氧基]-乙氧基}-乙氧基)-乙氧基]-乙氧基}-乙氧基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(1-(2-{2-[2-(4-甲基-4-甲基二硫基-戊醯基胺基)-乙氧基]-乙氧基}-乙氧基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(4-(2-{2-[2-(2-{2-[2-(4-甲基-4-甲基二硫基-戊醯基胺基)-乙氧基]-乙氧基}-乙氧基)-乙氧基]-乙氧基}-乙氧基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮] 8,8'-[(1-(2-[甲基-(2-甲基-2-甲基二硫基-丙基)-胺基]-乙氧基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(4-(3-[甲基-(4-甲基-4-甲基二硫基-戊醯基)-胺基]-丙基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(4-(3-[甲基-(2-甲基-2-甲基二硫基-丙基)-胺基]-丙基)-吡啶-2,6-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]8,8'-[(1-(4-甲基-4-甲基二硫基)-戊醯胺基)-苯-3,5-二甲基)-二氧基]-雙[(S)-2-亞乙-(E)-基-7-二甲氧基-1,2,3,11a-四氫-吡咯并[2,1-c][1,4]苯并二氮呯-5-酮]。 The combination of claim 26, wherein the cytotoxic agent is selected from the group consisting of a group of tomaymycin derivatives: 8,8'-[1,3-phenyldiylbis(methyleneoxy)]-bis[(S)-2-i-(E)-yl-7 -Methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'-[5- Methoxy-1,3-phenyldiylbis(methyleneoxy)]-bis[(S)-2-ethylidene-(E)-yl-7-methoxy-1,2,3,11a -tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'-[1,5-pentanediylbis(oxy)] -bis[(S)-2-ethylidene-(E)-yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4 Benzodiazepine-5-one]8,8'-[1,4-butanediylbis(oxy)]-bis[(S)-2-i-(E)-yl-7 -Methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'-[3- Methyl-1,5-pentanediylbis(oxy)]-bis[(S)-2-ethylidene-(E)-yl-7-methoxy-1,2,3,11a-tetra Hydrogen-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'-[2,6-pyridinediylbis(oxy)]-double [ (S)-2-Ethylene-(E)-yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo Diazin-5-one]8,8'-[4-(3-tert-butoxycarbonylaminopropoxy)-2,6-pyridinediylbis-(methyleneoxy)]-double [(S)-2-Ethylene-(E)-yl-7-methoxy-1, 2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'-[5-(3-Aminopropoxyl) ))-1,3-phenyldiylbis(methyleneoxy)]-bis[(S)-2-ethylidene-(E)-yl-7-methoxy-1,2,3,11a- Tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'-[5-(N-methyl-3-tributoxy Carbonylaminopropyl)-1,3-phenyldiylbis-(methyleneoxy)]-bis[(S)-2-ethylidene-(E)-yl-7-methoxy-1,2 ,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'-{5-[3-(4-methyl 4-methyldithiol (disulfanyl)-pentylamino)propoxy]-1,3-phenyldiylbis(methyleneoxy)}-bis[(S)-2-i- (E)-yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8 ,8'-[5-Ethylthiomethyl-1,3-phenyldiylbis(methyleneoxy)]-bis[(S)-2-A Methyl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] bis-{2 -[(S)-2-methylene-7-methoxy-5-oxooxy(oxo)-1,3,,11a-tetrahydro-5H-pyrrolo[2,1-c][1 , 4] benzodiazepine-8-yloxy]-ethyl}-carbamic acid tert-butyl ester 8,8'-[3-(2-ethylsulfonylthioethyl)-1,5 -Pentanediylbis(oxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1- c][1,4]benzodiazepine-5-one]8,8'-[5-(N-4-mercapto-4,4-dimethylbutanyl)amino-1,3-benzene Bis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4 Benzodiazepine-5-one]8,8'-[5-(N-4-methyldithio-4,4-dimethylbutanyl)-amino-1,3-phenyldiyl Bis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] Benzodiazepine-5-one]8,8'-[5-(N-methyl-N-(2-mercapto-2,2-dimethylethyl)amino-1,3-benzene (methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] Benzodiazepine-5-one]8,8'-[5-(N-methyl-N-(2-methyldithio-2,2-dimethylethyl)amino-1, 3-benzene Dikis (methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4 Benzodiazepine-5-one]8,8'-[(4-(2-(4-indolyl-4-methyl)-pentylamino-ethoxy)-pyridine-2,6- Dimethyl)-dioxy]-bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2, 1-c][1,4]benzodiazepine-5-one]8,8'-[(1-(2-(4-methyl-4-methyldithio)-pentanylamino) -ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-i-(E)-yl-7-dimethoxy-1,2, 3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] 8,8'-[(4-(3-(4-methyl-4-methyldithio)-pentylamino-propoxy)-pyridine-2,6-dimethyl)-dioxo ]]-bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1, 4]benzodiazepine-5-one]8,8'-[(4-(4-(4-methyl-4-methyldithio)-pentylamino-butoxy)-pyridine -2,6-dimethyl)-dioxy]-bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro- Pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'-[(4-(3-[4-(4-methyl-4-methyl) Thio-pentenyl)-piperazin-1-yl]-propyl)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)-2-i-(E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'- [(1-(3-[4-(4-Methyl-4-methyldithio-pentanyl)-piperazin-1-yl]-propyl)-benzene-3,5-dimethyl )-Dioxy]-bis[(S)-2-iB-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c ][1,4]benzodiazepine-5-one]8,8'-[(4-(2-{2-[2-(4-methyl-4-methyldithio-pentanyl) Amino)-ethoxy]-ethoxy}-ethoxy)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)-2-i-(E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyridyl咯[2,1-c][1,4]benzodiazepine-5-one]8,8'-[(1-(2-{2-[2-(2-{2-[2 -(4-methyl-4-methyldithio-pentanylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy )-Benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-i-(E)-yl-7-dimethoxy-1,2,3,11a- Tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'-[(1-(2-{2-[2-(4-methyl) 4-methyldithio-pentanylamino)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[( S)-2-Ethylene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine呯-5-ketone]8,8'-[(4-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldithio-pentanyl) Amino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-pyridine-2,6-dimethyl)-dioxy]- Bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzene And diazepine-5-one] 8,8'-[(1-(2-[Methyl-(2-methyl-2-methyldithio-propyl)-amino]-ethoxy)-benzene-3,5-di Methyl)-dioxy]-bis[(S)-2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1 -c][1,4]benzodiazepine-5-one]8,8'-[(4-(3-[methyl-(4-methyl-4-methyldithio-pentanyl) -amino]-propyl)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)-2-iethylene-(E)-yl-7-dimethoxy -1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]8,8'-[(4-(3-[A -(2-methyl-2-methyldithio-propyl)-amino]-propyl)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)- 2-ethylidene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine-5 -keto]8,8'-[(1-(4-methyl-4-methyldithio)-pentylamino)-benzene-3,5-dimethyl)-dioxy]-double [(S)-2-Ethylene-(E)-yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzo Diazin-5-one]. 如請求項26之結合物，其特徵在於該細胞毒性劑為選自由以下組成之群的細黴素衍生物：(2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-甲基硫基-乙基)-醯胺雙-[(2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-巰基乙基)-醯胺](2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-巰基-乙基)-醯胺(2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-甲基二硫基-乙基)-醯胺 (2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-甲基-2-甲基二硫基-丙基)-醯胺(2E,10E,12E,16Z,18E)-(R)-6-羥基-3,5,7,9,11,15,17-七甲基-19-((2S,3S)-3-甲基-6-側氧基-3,6-二氫-2H-吡喃-2-基)-8-側氧基-十九碳-2,10,12,16,18-五烯酸之(2-巰基-2-甲基-丙基)-醯胺。 A combination according to claim 26, characterized in that the cytotoxic agent is a leptomycin derivative selected from the group consisting of: (2E, 10E, 12E, 16Z, 18E)-(R)-6-hydroxy-3 ,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-oxy-3,6-dihydro-2H-pyran-2 -yl)-8-sideoxy-nine-carbon-2,10,12,16,18-pentenoic acid (2-methylsulfanyl-ethyl)-nonylamine bis-[(2E, 10E, 12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptyl-19-((2S,3S)-3-methyl-6-side Oxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy-nine-carbon-2,10,12,16,18-pentenoic acid (2-mercaptoethyl) )-decylamine](2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptyl-19-((2S,3S) )-3-methyl-6-tertiaryoxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy-nine carbon-2,10,12,16,18- (2-mercapto-ethyl)-decylamine (2E, 10E, 12E, 16Z, 18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-seven Methyl-19-((2S,3S)-3-methyl-6-oxooxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy-nine carbon- (2-methyldithio-ethyl)-decylamine 2,10,12,16,18-pentenoic acid (2E, 10E, 12E, 16Z, 18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptyl-19-((2S,3S)-3-A Keto-6-o-oxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy-nine-carbon-2,10,12,16,18-pentenoic acid 2-methyl-2-methyldithio-propyl)-decylamine (2E, 10E, 12E, 16Z, 18E)-(R)-6-hydroxy-3,5,7,9,11,15 ,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxooxy-3,6-dihydro-2H-pyran-2-yl)-8-sideoxy- (2-nonyl-2-methyl-propyl)-guanamine of nineteen carbon-2,10,12,16,18-pentenoic acid. 如請求項1-2、6-13及15-24中任一項之抗體或其抗原決定基結合片段或如請求項25-30中任一項之結合物，其係用作藥劑。 The antibody or its epitope binding fragment of any one of claims 1-2, 6-13 and 15-24, or a combination according to any one of claims 25-30, for use as a medicament. 一種醫藥組合物，其含有如請求項1-24中任一項之抗體或其抗原決定基結合片段或如請求項25-30中任一項之結合物及醫藥學上可接受之載劑或賦形劑。 A pharmaceutical composition comprising the antibody of any one of claims 1 to 24, or an antigenic determinant binding fragment thereof, or a combination according to any one of claims 25-30, and a pharmaceutically acceptable carrier or excipient. 如請求項32之醫藥組合物，其特徵在於該組合物含有另一種治療劑。 A pharmaceutical composition according to claim 32, characterized in that the composition contains another therapeutic agent. 如請求項33之醫藥組合物，其特徵在於該另一種治療劑為表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、肝細胞生長因子(HGF)、組織因子(TF)、蛋白質C、蛋白質S、血小板衍生之生長因子(PDGF)、神經調節素-1(heregulin)、巨噬細胞刺激蛋白(MSP)或血管內皮生長因子(VEGF)之拮抗劑；或表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、肝細胞生長因子(HGF)、組織因子(TF)、蛋白質C、蛋白質S、血小板衍生之生長因子(PDGF)、神經調節素-1、巨噬細胞刺激蛋白(MSP)或血管內皮生長因子(VEGF)之受體(包括HER2受體、HER3受體、c-MET及其他受體酪胺酸激酶)之拮抗劑。 The pharmaceutical composition according to claim 33, characterized in that the other therapeutic agent is epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C , protein S, platelet-derived growth factor (PDGF), neuromodulin-1 (heregulin), macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF) antagonist; or epidermal growth factor (EGF), Fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), neurotonin-1, macrophage stimulating protein ( Antagonists of MSP) or vascular endothelial growth factor (VEGF) receptors, including the HER2 receptor, HER3 receptor, c-MET, and other receptor tyrosine kinases. 如請求項33之醫藥組合物，其特徵在於該另一種治療劑為一種對抗一群選自包含下列之分化抗原之抗體：CD3、CD14、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD36、 CD40、CD44、CD52、CD55、CD59、CD56、CD70、CD79、CD80、CD103、CD134、CD137、CD138及CD152。 The pharmaceutical composition of claim 33, wherein the another therapeutic agent is an antibody against a population selected from the group consisting of: CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36 , CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138 and CD152. 一種如請求項1-24中任一項之抗體或其抗原決定基結合片段或如請求項25-30中任一項之結合物之用途，其係用於製造用於治療癌症或自體免疫疾病之藥劑。 The use of an antibody of any one of claims 1 to 24, or an antigenic determinant-binding fragment thereof, or a combination according to any one of claims 25 to 30, for use in the manufacture of a medicament for the treatment of cancer or autoimmunity The agent of disease. 如請求項36之用途，其特徵在於該癌症係選自由以下組成之群：癌瘤，包括膀胱、乳房、結腸、腎、肝、肺、卵巢、胰腺、胃、子宮頸、甲狀腺及皮膚之癌瘤；包括鱗狀細胞癌；間葉源性腫瘤，包括纖維肉瘤及橫紋肌肉瘤；其他腫瘤，包括黑素瘤、精原細胞瘤、畸胎癌、神經母細胞瘤及神經膠質瘤；中樞及周邊神經系統之腫瘤，包括星形細胞瘤、神經母細胞瘤、神經膠質瘤及神經鞘瘤；間葉源性腫瘤，包括纖維肉瘤、橫紋肌肉瘤及骨肉瘤；及其他腫瘤，包括黑素瘤、著色性乾皮病、角化棘皮瘤、精原細胞瘤、甲狀腺濾泡癌及畸胎癌。 The use of claim 36, characterized in that the cancer is selected from the group consisting of cancer, including bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin cancer Tumors; including squamous cell carcinoma; mesenchymal tumors, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; central and peripheral Nervous system tumors, including astrocytoma, neuroblastoma, glioma, and schwannomas; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, coloration Dry skin disease, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma. 如請求項36之用途，其特徵在於該癌症為淋巴系之造血腫瘤，包括白血病、非霍奇金氏淋巴瘤、急性淋巴細胞性白血病、急性淋巴母細胞白血病、B細胞淋巴瘤、T細胞淋巴瘤、伯基特氏淋巴瘤、霍奇金氏淋巴瘤、毛細胞白血病、多發性骨髓瘤、慢性淋巴細胞性白血病；骨髓系之造血腫瘤，包括急性及慢性骨髓性白血病及前髓細胞性白血病。 The use of claim 36, characterized in that the cancer is a hematopoietic tumor of the lymphoid system, including leukemia, non-Hodgkin's lymphoma, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma Tumor, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, multiple myeloma, chronic lymphocytic leukemia; hematopoietic tumors of the myeloid lineage, including acute and chronic myeloid leukemia and promyelocytic leukemia . 如請求項36之用途，其特徵在於該癌症為多發性骨髓瘤。 The use of claim 36, characterized in that the cancer is multiple myeloma. 如請求項36之用途，其特徵在於該自體免疫或發炎疾病係選自由以下組成之群：全身性紅斑性狼瘡症、類風濕性關節炎、多發性硬化症、克隆氏病(Crohn's disease)、潰瘍性結腸炎、胃炎、橋本氏甲狀腺炎(Hashimoto's thyroiditis)、強直性脊椎炎、C型肝炎相關之冷凝球蛋白性(cryoglobulinemic)血管炎、慢性局竈 性腦炎、大疱性類天疱瘡、A型血友病、膜性增生性絲球體腎炎、修格蘭氏症候群(Sjogren's syndrome)、成人及青少年皮肌炎、成人多發性肌炎、慢性蕁麻疹、原發性膽汁性肝硬化症、特發性血小板減少性紫癜、視神經脊髓炎、格雷氏甲狀腺機能障礙疾病(Graves' dysthyroid disease)、大疱性類天疱瘡、膜性增生性絲球體腎炎、徹奇-斯全司症候群(Churg-Strauss syndrome)及哮喘。 The use of claim 36, characterized in that the autoimmune or inflammatory disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Crohn's disease , ulcerative colitis, gastritis, Hashimoto's thyroiditis, ankylosing spondylitis, hepatitis C-related condensed globulin (cyoglobulinemic) vasculitis, chronic focal Encephalitis, bullous pemphigoid, hemophilia A, membranous proliferative spheroid nephritis, Sjogren's syndrome, adult and adolescent dermatomyositis, adult polymyositis, chronic sputum Measles, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, optic neuromyelitis, Graves' dysthyroid disease, bullous pemphigoid, membranous proliferative glomerulonephritis , Churg-Strauss syndrome and asthma. 如請求項36之用途，其進一步包含另一種治療劑用於製造相同或不同組合物之用途。 The use of claim 36, which further comprises the use of another therapeutic agent for the manufacture of the same or different compositions. 如請求項41之用途，其特徵在於該另一種治療劑為纖維母細胞生長因子(FGF)、肝細胞生長因子(HGF)、組織因子(TF)、蛋白質C、蛋白質S、血小板衍生之生長因子(PDGF)、神經調節素-1(heregulin)、巨噬細胞刺激蛋白(MSP)或血管內皮生長因子(VEGF)之拮抗劑；或表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、肝細胞生長因子(HGF)、組織因子(TF)、蛋白質C、蛋白質S、血小板衍生之生長因子(PDGF)、神經調節素-1、巨噬細胞刺激蛋白(MSP)或血管內皮生長因子(VEGF)之受體(包括HER2受體、HER3受體、c-MET及其他受體酪胺酸激酶)之拮抗劑。 The use of claim 41, characterized in that the other therapeutic agent is fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor. (PDGF), neuromodulin-1 (heregulin), macrophage stimulating protein (MSP) or an antagonist of vascular endothelial growth factor (VEGF); or epidermal growth factor (EGF), fibroblast growth factor (FGF), Hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), neurotonin-1, macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF) Antagonists of receptors (including HER2 receptor, HER3 receptor, c-MET, and other receptor tyrosine kinases). 如請求項41之用途，其特徵在於該另一種治療劑為一種對抗一群選自下列之分化抗原之抗體：CD3、CD14、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD36、CD40、CD44、CD52、CD55、CD59、CD56、CD70、CD79、CD80、CD103、CD134、CD137、CD138及CD152。 The use of claim 41, characterized in that the other therapeutic agent is an antibody against a population of differentiation antigens selected from the group consisting of CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138 and CD152. 一種診斷已知或懷疑患有表現CD38之癌症之個體中之表現CD38之癌症的活體外方法，該方法包含：a)使該患者之細胞與如請求項1-24之抗體或其抗原決定基結合 片段接觸，b)測量該抗體或其抗原決定基結合片段與該等細胞之結合，及c)將部分(b)中之表現與正常參考個體或標準之表現比較。 An in vitro method for diagnosing a cancer exhibiting CD38 in an individual known or suspected to have a cancer exhibiting CD38, the method comprising: a) subjecting the patient's cells to an antibody or epitope thereof as claimed in claims 1-24 Combine Fragment contact, b) measuring the binding of the antibody or its epitope binding fragment to the cells, and c) comparing the performance in part (b) with the performance of a normal reference individual or standard. 如請求項44之方法，其特徵在於該癌症係選自由以下組成之群：癌瘤，包括膀胱、乳房、結腸、腎、肝、肺、卵巢、胰腺、胃、子宮頸、甲狀腺及皮膚之癌瘤；包括鱗狀細胞癌；間葉源性腫瘤，包括纖維肉瘤及橫紋肌肉瘤；其他腫瘤，包括黑素瘤、精原細胞瘤、畸胎癌、神經母細胞瘤及神經膠質瘤；中樞及周邊神經系統之腫瘤，包括星形細胞瘤、神經母細胞瘤、神經膠質瘤及神經鞘瘤；間葉源性腫瘤，包括纖維肉瘤、橫紋肌肉瘤及骨肉瘤；及其他腫瘤，包括黑素瘤、著色性乾皮病、角化棘皮瘤、精原細胞瘤、甲狀腺濾泡癌及畸胎癌。 The method of claim 44, wherein the cancer is selected from the group consisting of cancer, including bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, and skin cancer Tumors; including squamous cell carcinoma; mesenchymal tumors, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; central and peripheral Nervous system tumors, including astrocytoma, neuroblastoma, glioma, and schwannomas; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, coloration Dry skin disease, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma. 如請求項44之方法，其中該癌症為淋巴系之造血腫瘤，包括白血病、非霍奇金氏淋巴瘤、急性淋巴細胞性白血病、急性淋巴母細胞白血病、B細胞淋巴瘤、T細胞淋巴瘤、伯基特氏淋巴瘤、霍奇金氏淋巴瘤、毛細胞白血病、多發性骨髓瘤、慢性淋巴細胞性白血病；骨髓系之造血腫瘤，包括急性及慢性骨髓性白血病及前髓細胞性白血病。 The method of claim 44, wherein the cancer is a lymphoid hematopoietic tumor, including leukemia, non-Hodgkin's lymphoma, acute lymphocytic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, multiple myeloma, chronic lymphocytic leukemia; hematopoietic tumors of the myeloid lineage, including acute and chronic myeloid leukemia and promyelocytic leukemia. 如請求項44之方法，其特徵在於該等細胞為來自該患者之經冷凍或固定之組織或細胞。 The method of claim 44, wherein the cells are frozen or fixed tissues or cells from the patient. 一種聚核苷酸，其編碼一種選自由SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、38、40、44、46、48、50、52、56、58、60、68、70及72組成之群之多肽。 A polynucleotide encoding one selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 40, 44, 46, 48, 50, 52, 56, 58, 60, 68, 70 and 72 a group of polypeptides. 如請求項48之聚核苷酸，其特徵在於該聚核苷酸具有一個序列與一個選自由SEQ ID NO：37、39、43、45、47、49、51、55、 57、59、67、69及71組成之群之聚核苷酸享有至少80%同源性。 The polynucleotide of claim 48, characterized in that the polynucleotide has a sequence and one selected from the group consisting of SEQ ID NOs: 37, 39, 43, 45, 47, 49, 51, 55, Polynucleotides of the group consisting of 57, 59, 67, 69 and 71 possess at least 80% homology. 一種重組載體，其包含如請求項48或49中任一項之核酸。 A recombinant vector comprising the nucleic acid of any one of claims 48 or 49. 一種宿主細胞，其包含如請求項50之載體。 A host cell comprising the vector of claim 50.
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